ES2311731T3 - PROTEINS OF PESTICIDE ACTION AND POLYNUCLEOTIDES THAT CAN BE OBTAINED FROM PAENIBACILLUS SPECIES. - Google Patents

Abstract

The subject invention provides unique biological alternatives for pest control. More specifically, the present invention relates to novel pesticidal proteins, polynucleotides that encode such toxins, and to methods of toxins to control insects and other plant pests. The subject invention relates to the surprising discovery that Paenibacillus species, and proteins therefrom, have toxicity to lepidopterans. There have been no known reports of a Paenibacillus species, strain, or protein having toxicity to lepidopterans. This is also the first known example of a Paenibacillus Cry protein that is toxic to lepidopterans. Furthermore, this is the first known report of Paenibacillus having TC-like proteins. The DAS 1529 isolate disclosed here is also the first known example of a natural bacterium that produces both a Cry toxin and toxin complex (TC) proteins. The subject invention also relates to new classes of Cry and TC proteins that are pesticidally active.

Description

Pesticide and polynucleotide action proteins that can be obtained from Paenibacillus species.

Cross reference to related requests

This request claims priority to the provisional application Serial No. 60 / 392,633, filed on 28 June 2002, and provisional application Serial No. 60 / 441,647, filed on January 21, 2003.

Background of the invention

Insects and pests cost farmers billions of dollars annually in crop losses and in maintenance costs of these pests under control. The losses caused by insect pests in production environments agricultural include declines in crop yields, reduction in crop quality, and increased costs of The recollection. Insect pests are also a burden for vegetable and fruit growers, to flower growers ornamentals and home gardeners and homeowners House.

Cultivation procedures, such as a crop rotation and the application of high levels of nitrogen fertilizers, have partially directed the problems caused by agricultural pests. However, the economic demands of the use of cultivated land restrict the use of crop rotation. In addition, the hibernation characteristics of some insects disrupt the crop rotations in some areas.

Thus, chemical insecticides synthetics rely to the greatest extent on achieving a sufficient level of control. However, the use of synthetic chemical insecticides It may have several drawbacks. For example, the use of some of these chemical compounds can adversely affect the beneficial insects Target insects have also developed resistance to some chemical pesticides. This has been alleviated. partially through various management strategies to the resistance, but there is a growing need for agents Pest control alternatives. In addition, large populations of weevils, heavy rains, and improper calibration of equipment insecticide application may result in poor control. Inappropriate use of insecticides reaches issues environmental factors such as soil pollution and both surface water supplies as underground. Waste they can also remain in treated fruits, vegetables and Other treated plants. Working with some insecticides too It can pose risks for the people who apply them. For the therefore, synthetic chemical insecticides are being examined for increasingly because of its potentially environmental consequences Toxic The new rigorous restrictions on the use of pesticides and the elimination of some effective pesticides from the market can limit economic and effective options for the control of harmful and expensive pests.

Due to the problems associated with the use of synthetic chemical pesticides, there is an obvious need for limit the use of these agents and a need to identify alternative control agents. Pesticide replacement synthetic chemicals, or combinations of these agents with biological pesticides, can reduce compound levels Toxic chemicals in the environment.

Some biological pesticide agents that are now being used with some success are derived from soil microbes Bacillus thuringiensis ( Bt .). The soil microbe Bacillus thuringiensis ( Bt .) Is a Gram-positive spore-forming bacterium. Most strains of Bt . Do not show pesticidal activity. Some strains of Bt produce, and can be characterized by, parasporal crystalline protein inclusions. These inclusions often appear microscopically as characteristic shaped crystals. Some Bt proteins. They are highly toxic to pests, such as insects, and are specific in their toxic activity. Certain B proteins. t . They are associated with inclusions. These "δ-endotoxins" are different from exotoxins, which have a non-specific host range. Other species of Bacillus also produce pesticidal proteins.

Certain Bacillus toxin genes have been isolated and sequenced, and recombinant DNA-based products have been produced and approved for use. In addition, with the use of genetic engineering techniques, various approaches to administer these toxins to agricultural environments are being perfected. These include the use of genetically engineered plants with toxin genes for insect resistance and the use of stabilized intact microbial cells as toxin distribution vehicles. In this way, the toxin genes isolated from Bacillus become commercially valuable.

\hbox{endotoxina que es tóxica para  las
larvas de numerosos insectos de lepidópteros.}

Commercial use of Bt pesticides. It was initially restricted to the direction of a narrow range of lepidopteran (caterpillar) pests. Preparations of the spores and crystals of B. thuringiensis subspecies kurstaki have been used for many years as commercial insecticides for lepidopteran pests. For example, B. thuringiensis var. Kurstaki HD-1 produces a δ

 \ hbox {endotoxin that is toxic to
larvae of numerous insects of lepidoptera.} 

More recently, new subspecies of Bt have been identified, and genes responsible for active δ-endotoxin proteins have been isolated. Höfte and Whiteley classified the genes of the B crystalline protein. t . in four main classes (Höfte, H., HR Whiteley [1989] Microbiological Reviews 52 (2): 242-255). The classes were Cry I (specific to Lepidoptera), Cry II (specific to Lepidoptera and Diptera), Cry III (specific to Coleoptera), and Cry IV (specific to Diptera). The discovery of specifically toxic strains for other pests has been reported. For example, Cry V and Cry VI were proposed to designate a class of toxin genes that are specific to nematodes.

Cry I crystalline proteins specific to lepidoptera, in their natural state, proteins of approximately 130 to 140 kDa, which accumulate in bipyramidal crystalline inclusions during sporulation of B. thuringiensis . These proteins are protoxins that are solubilized in the alkaline environment of the midgut of insects and are proteolytically converted by crystal-associated proteases or from the midgut of larvae into a fragment of the toxic nucleus of 60 to 70 kDa. This activation can also be carried out in vitro with a variety of proteases. The toxic domain is located in the N-terminal half of protoxin. This was demonstrated for the Cry IA (b) and Cry IC proteins by N-terminal amino acid sequencing of the trypsin-activated toxin. Höfte et al . 1989. The cleavage occurs at the C-terminal end of a conserved region called "Block 5", which thus forms the C-terminus of the nucleus toxin. A short segment of N-terminal protoxin can also be processed outside. The N-terminal cleavage site is also highly conserved for Cry IA and Cry ID proteins, which suggests that these proteins, the N-terminus of the toxic fragment is located in the same position. However, Cry IB, is different from the other Cry I proteins in this region. It has been known if this protein is also processed at the N end. Höfte et al . 1989

Suppression analysis of several cry I genes further confirmed that the 3 'half of protoxin is not required for toxic activity. One of the shortest fragments reviewed was located between codons 29 and 607 for Cry IAb. In addition, the withdrawal of four codons from the 3 'end or eight codons from the 5' end codons completely suppressed the toxic activity of the gene product. Similar observations were made for the cry IA (a) and cry LA (c) genes. Höfte et al . 1989

The cry II genes encode 65 kDa proteins that form cube-like inclusions in strains of various subspecies. These crystalline proteins were previously designated "P2" proteins, as opposed to the 130 kDa P1 crystalline proteins present in the same strains. Höfte et al . 1989

An A cry ILA gene was cloned from B. thuringiensis subsp. Kurstaki HD- 263 and expressed in Bacillus megaterium . The cells that produce the Cry IIA protein were toxic to the Heliothis virescens and Lymantria dispar lepidoptera species as well as to the larvae of the Aedes aegypti dipterans . Widner and Whitely (1989, J. Bacteriol. 171: 965-974) two related cloned genes ( cry IIA and cry IIB) of B. thuringgiensis subsp. Kurstaki HD-1. Both genes encode 633 amino acid proteins with a predicted molecular mass of 71 kDa (slightly greater than the apparent molecular mass determined for P2 proteins produced in B. thuringieiensis ). Although Cry IIA and Cry IIB proteins are highly homologous (~ 87% amino acid identity), they differ in the insecticidal spectrum. Cry IIA is active against both a species of lepidoptera ( Manduca sixth ) and a diptera ( Aedes aegypti ), while cry IIB is toxic only to lepidopteran insects. Höfte et al. 1989. Cry II toxins, as a group, tend to be relatively more conserved at the sequence level (> 80% identity) than other groups. On the contrary, there are many Cry I toxins, for example, including some that are less than 60% identical.

Höfte and Whiteley's 1989 nomenclature and classification scheme for crystalline proteins was based on both the deduced amino acid sequence and the host range of the toxin. That system was adapted to cover 14 different types of toxin genes that were divided into five main classes. The nomenclature scheme of 1989 could not be used since more and more genes were discovered that encoded proteins with variable spectra of pesticidal activity. Thus, a revised nomenclature scheme was adopted, which is based solely on amino acid identity (Crickmore et al ., 1998, Microbiology and Molecular Biology Reviews 62: 807-813). The mnemonic code " cry " has been maintained for all toxin genes except cytA and cytB, which maintains a separate class. Roman numerals have been changed to Arabic numbers in the primary category, and parentheses in the tertiary category have been removed. Many of the original names have remained, with the exceptions indicated, although numerous have been re-classified. There are now at least 37 primary classes of Cry proteins, and two primary classes of cyt toxins. Other types of toxins, such as those of WO 98/18932 and WO 97/40162, have been discovered from B. thuringiensis.

There are some obstacles to the successful agricultural use of Bacillus pesticide proteins (and other biological species). Certain insects can be refractory to Bacillus toxins. Insects such as the crickets of the cotton capsule, black worm, and Helicoverpa zea , as well as adult insects of most species, have so far shown no significant sensitivity to many δ-endotoxins of Bt .

Another potential obstacle is the development of resistance to Bt toxins by insects. Bt protein toxins were initially formulated as spraying insect control agents. A more recent application of B technology. t . It has been isolating and transforming plants with genes that encode these toxins. The transgenic plants subsequently produce these toxins, thereby providing insect control. See U.S. Patents Nos. 5,380,831; 5,567,600; and 5,567,862 from Mycogen Corporation. The plants of B. t . GM crops are completely effective, and use is predicted to be high in some crops and areas. This has caused some interest in resistance management issues to arise more quickly than with sprayable applications. Although numerous insects have been selected for resistance to Bt toxins in the laboratory, only the diamondback moth ( Plutella xylostella ) has demonstrated resistance in an established field (Ferre, J. and Van Rie, J., Annu. Rev. Entomol. 47: 501-533, 2002).

Resistance management strategies in the technology of transgenic plants in B. t. they have become of great interest (for example, as in a natural bacterium, several multiple toxins can be exposed in the same plant, thereby greatly reducing the possibility that an insect that could be resistant to a toxin would survive for expand resistance). Various strategies have been suggested to conserve the ability to use B. thuringiensis toxins effectively. These strategies include high dose with protection, and alternating with, or joint deployment of, different toxins (McGaughey et al . (1998), "Bt Resistance Management", Nature Biotechnol 16: 144-146).

Thus, there is a great need to develop additional genes that can be expressed in plants in order to effectively control various insects. In addition to continually trying to discover new Bt toxins, it would be completely desirable to discover other bacterial sources (other than Bt .) That produce toxins that could be used in transgenic plant strategies, or that could be combined with B. t .s to produce transgenic plants that control insects.

Recent efforts to clone insecticidal toxin genes from the Photorhabdus / Xenorhabdus bacteria group present potential alternatives to toxins derived from B. thuringiensis . It has been known in the art that bacteria of the genus Xenorhabdus are symbioticly associated with the Steinernema nematode. Unfortunately, as indicated in numerous articles, bacteria only have pesticidal activity when injected into insect larvae and showed no biological activity when administered orally.

It has been difficult to effectively exploit the insecticidal properties of the nematode its symbiotic bacteria. Thus, it would be totally desirable to discover proteinaceous agents of the Xenorhabdus bacteria that have oral activity so that the products produced from them could be formulated in the form of a sprayable insecticide, or the bacterial genes encoding said proteinaceous agents can be isolated. and use in the production of transgenic plants. WO 95/00647 refers to the use of Xenorhabdus protein toxin to control insects, but does not recognize active toxins by mouth. WO 98/08388 refers to pesticide agents administered orally from Xenorhabdus . US Patent No. 6,048,838 refers to protein toxin / toxin complexes, which have oral activity, which can be obtained from Xenorhabdus species and strains.

Photorhabdus and Xenorhabdus spp. Gram-negative bacteria that are entomopathogenic and symbiotic are associated with soil nematodes. These bacteria are found in the intestine of entomopathogenic nematodes that invade and kill insects. When the nematode invades an insect host, the bacteria are released into the insect hemocoel (the open circulatory system), and both the bacteria and the nematodes subjected to multiple rounds of replication; the insect host typically dies. These bacteria can be grown outside the nematode hosts. For a more detailed description of these bacteria, see Forst and Nealson, 60 Microbiol. Rev. 1 (1996), p. 21-43.

The genus Xenorhabdus is defined taxonomically as a member of the Enterobacteriaceae family, although it has certain atypical characters of this family. For example, strains of this genus are typically negative for the reduction of nitrates and catalase negative. Xenorhabdus has been subdivided only recently to create a second genus, Photorhabdus , which comprises individual species Photorhabdus luminescens ( previously Xenorhabdus luminescens ) (Boemare et al ., 1993 Int. J. Syst. Bacteriol. 43, 249-255). This differentiation is based on several distinctive features that are easily identified by those skilled in the art. These differences include the following: DNA-DNA characterization studies; presence ( Photorhabdus ) or phenotypic absence ( Xenorhabdus ) of catalytic activity; presence ( Photorhabdus ) or absence ( Xenorhabdus ) of bioluminescence; the Nematode host family in which Xenorhabdus is found in Steinernematidae and Photorhabdus pass it in Heterorhabditidae ); as well as in comparison, analysis of cellular fatty acids (Janse et al . 1990, Lett. Appl. Microbiol. 10, 131-135; Suzuki et al . 1990, J. Gen. Appl. Microbiol., 36, 393-401) . In addition, recent centralized molecular studies in sequence analysis (Rainey et al . 1995, Int. J. Syst. Bacteriol., 45, 379-381) and restriction (Brunel et al ., 1997, App. Environ. Micro., 63, 574-580) of the 16S rRNA genes also support the separation of these two genera.

The expected characters for Xenorhabdus are the following: Gram negative rods, yellow / brown pigmentation colony, presence of inclusion bodies, absence of catalase, inability to reduce nitrates, absence of bioluminescence, ability to accept medium dyes, positive gelatin hydrolysis , growth in Enterobacteriaceae selective media, growth below a temperature of 37 ° C, survival under anaerobic conditions, and motility.

Currently, the bacterial genus Xenorhabdus comprises four recognized species, Xenorhabdus nematophilus , Xenorhabdus poinarii , Xenorhabdus bovienii and Xenorhabdus beddingii (Brunel et al ., 1997, App. Environ. Micro., 63, 574-580). A variety of related strains have been described in the literature ( eg, Akhurst and Boemare 1988 J. Gen. Microbiol., 134, 1835-1845; Boemare et al . 1993 Int. J. Syst. Bacteriol. 43, p. 249 - 255; Putz et al . 1990, Appl. Environ. Microbiol., 56, 181-186, Brunel et al ., 1997, App. Environ. Micro., 63, 574-580, Rainey et al . 1995, Int J. Syst. Bacteriol., 45, 379-381).

The bacteria Xenorhabdus and Photorhabdus secrete a wide diversity of substances in the culture medium; These secretions include lipases, protease, antibiotics and lipopolysaccharides. The purification of different protease fractions has been clearly demonstrated that they are not involved in the oral toxic activity of the culture medium of P. luminescens (which has subsequently been determined to reside with Tc proteins only). Several of these substances have been previously involved in insect toxicity but until recently no insecticidal gene had been cloned. However, purification and separation of proteases will also facilitate an examination of their assumed role in, for example, inhibition of antibacterial proteins such as cecropin. RH ffrench-Constant and Bowen, Current Opinions in Micriobiology, 1999, 12: 284-288. See RH ffrench-Constant et al . 66 AEM No. 8, p. 3310-3329 (August 2000), for a review of the various factors involved in the virulence of Photorhabdus of insects.

There has been substantial progress in the cloning of genes encoding insecticidal toxins from both Photorhabdus luminescens and Xenorhabdus nematophilus . The toxin complex that encodes the P. luminescens genes were examined first. See, for example, WO 98/08932. The "Parallel" genes were more recently cloned from X. nematophilus Morgan et al ., Applied and Environmental Microbiology 2001, 67: 2062-69.

Four different toxin complexes (TCs) -Tca, Tcb, Tcc and Tcd have been identified in Photorhabdus spp. Each of these toxin complexes resolves both individual and dimeric species in a native agarose gel but the resolution in a denaturing gel reveals that each complex consists of a range of species between 25-280 kDa. The ORFs that encode Photorhabdus CT scans , in conjunction with protease cleavage sites (vertical arrows), are illustrated in Figure 1. See also RH ffrench-Constant and Bowen, 57 Cell. Mol. Life Sci. 828-833 (2000).

The genomic libraries of P. luminescens were selected with DNA probes and with monoclonal and / or polyclonal antibodies induced against toxins. Four tc loci were cloned: tca, tcb , tcc and tcd . The tca locus is an assumed operon of three open reading frames (ORFs), tcaA , tcaB , and tcaC transcribed from the same strand of DNA, with a smaller terminal ORF ( tcaZ ) that is transcribed in the opposite direction. The tcc locus also comprises three ORFs supposedly transcribed in the same direction ( tccA , tccB , and tccC ). The tcb locus is a single large ORF ( tcbA ), and the tcd locus is composed of two ORFs ( tcdA and tcdB ); tcbA and tcdA , each approximately 7.5 kb, encode large insect toxins. TcdB has homology with TcaC. Many of these gene products were determined to be cleaved by proteases. For example, both TcbA and TcdA are cleaved into three fragments called i, ii and iii (for example, TcbAi, TcbAii and TcbAiii). The products of the tca and tcc ORFs are also cleaved. See Figure 1. See also RH ffrench-Constant and DJ Bowen, Current Opinions in Microbiology, 1999, 12: 284-288.

Bioassays of the Tca toxin complexes revealed that they were highly toxic to the first-stage tomato horned worm ( Manduca sixth ) when given orally (LD50 of 875 ng per square centimeter of artificial diet). RH ffrench-Constant and Bowen 1999.

Feeding was inhibited at doses of Tca as low as 40 ng / cm2. given the predicted high molecular weight of Tca, on a molar basis, the toxins of P. Luminescens are highly active and relatively few molecules appear to be necessary to exert a toxic effect RH ffrench-Constant and Bowen, Current Opinions in Micriobiology, 1999, 12: 284-288.

None of the four loci showed similarity global with all known function sequences in GenBank. Sequence similarity regions raised some suggestion that these proteins (TcaC and TccA) can overcome the immunity of insects by attacking insect hemocytes. RH. ffrench-Constant and Bowen, Current Opinions in Microbiology, 1999, 12: 284-288.

\hbox{Constant
y Bowen, Current Opinions  in Microbiology, 1999, 12: 284 -
288.}

TcaB, TcbA, and TcdA all show the conservation of amino acids (~ 50% identity), compared to each other, immediately around their predicted protease cleavage sites. This conservation between three different TC proteins that suggest that all may be processed by the same or similar proteases. TcbA and TcdA also share? 50% of global identity, as well as a similar predicted pattern of both carboxy- and amino-terminal cleavage. It is postulated that these proteins could thus be homologous to others. In addition, the similar, large size of TcbA and TcdA, and also the fact that both toxins appear to act on the gut of the insect, may suggest similar modes of action. RH ffrench-

 \ hbox {Constant
and Bowen, Current Opinions in Microbiology, 1999, 12: 284 -
288.} 

Genetic suppression / inactivation studies suggest that the products of the loci tca and tcd loci are responsible for oral toxicity to lepidoptera. The suppression of any of the tca or tcd genes greatly reduced oral activity against Manduca sixth . That is, the products of the loci tca and tcd loci are oral toxins for lepidoptera themselves; Its combined effect contributed to the majority of oral secreted activity. RH ffrench-Constant and DJ Bowen, 57 Cell. Mol. Life Sci. 831 (2000). Interestingly, the deletion of any of the tcb or tcc loci alone also reduces mortality, which suggests that complex interactions exist between different gene products. In this way, the products of the tca locus can enhance the toxicity of the tcd products. Alternatively, tcd products can modulate the toxicity of tca products and possibly other complexes. It should be noted that the above is related to oral activity against a single species of insect, tcb or tcc loci can produce toxins that are more active against groups of insects (or active by injection directly into the hemocoel pathway of insects). normal administration when secreted by bacteria in vivo ). RH ffrench- Constant and Bowen, Current Opinions in Microbiology, 1999, 12: 284-288.

WO 01/11029 describes sequences of nucleotides that encode TcdA and TcbA and have compositions of base that have been altered from the native genes to make them more similar to plant genes. Plants are also described GMOs that express Toxin A and Toxin B.

Of the isolated toxins isolated from Photorhabdus luminescens (W-14), the so-called Toxin A and Toxin B have been the subject of centralized research for their activity against target insect species of interest ( for example, the corn root worm ). Toxin A comprises two different subunits. The native tcdA gene encodes the protoxin TcdA. As determined by mass spectrometry, TcdA is processed by one or more proteases to provide Toxin A. More specifically, TcdA is a protein of approximately 282.9 kDa (2516 aa) that is processed to provide TcdAi (the first 88 amino acids ), TcdAii (the following 1849 aa; a protein of approximately 208.2 kDa encoded by nucleotides 265-5811 of tcdA ), and TcdAiii, a protein of approximately 63.5 kDa (579 aa) (encoded by nucleotides 5812 - 7551 of tcdA ). TcdAii and TcdAiii seem to be assembled in a dimer (perhaps aided by TcdAi), and the dimers are assembled in a four-dimer tetramer. Toxin B is similarly derived from TcbA.

Although the exact molecular interactions of the TC proteins with each other, and their mechanism (s) of action, are not currently understood, it is known, for example, that the Photorhabdus Tca toxin complex is toxic to Manduca sixth . In addition, some TC proteins are known to have "autonomous" insecticidal activity, while other CT proteins are known to enhance or increase the activity of autonomous toxins. It is known that the TcdA protein is active, alone, against Manduca sixth, but that TcdB and TccC, together, can be used to enhance the activity of TcdA. Waterfield, N. et al ., Appl. Environ. Microbiol 2001, 67: 5017-5024. TcbA (there is only one Tcb protein) is another autonomous Photorhabdus toxin. The activity of this toxin (TcbA) can also be enhanced by TcdB in conjunction with TccC type proteins.

US Patent Application 20020078478 provides nucleotide sequences for two enhancer genes, tcdB2 and tccC2, of the genomic tcd region of Photorhabdus luminescens W-14. It is known in this document that the coexpression of tcdB and tccC1 with tcdA results in enhanced levels of oral insect toxicity compared to that obtained when tcdA expresses itself. Joint expression of tcdB and tccC1 with tcdA or tcbA provides enhanced oral insect activity.

As indicated in the Table below, TccA has a level of homology with the N-terminus of TcdA, and TccB has some level of homology with the C-terminus of TcdA. TccA and TccB are much less active on certain test insects than is TcdA. TccA and TccB of the Photorhabdus W-14 strain are called "Toxin D". "Toxin A" (TcdA), "Toxin B" (TcbA), and "Toxin C" (TcaA and TcaB) are also indicated below. In addition, TcaA has some level of homology with TccA and similarly with the N-terminus of TcdA. Even in addition, TcaB has some level of homology with TccB and similarly with the N end of TcdA. TccA and TcaA are of similar size, as are TccB and TcaB. TcdB has a significant level of similarity (both in sequence and in size) to TcaC.

one

The epithelium of the middle intestine of insects contains both column-shaped (structural) and cup-shaped (secretory) cells. Ingestion of tca products by M. sixth leads to apical ingestion of and formation of bubbles of large cytoplasmic vesicles by Column-shaped cells, which lead to the eventual extrusion of nuclei of cells into vesicles in the lumen of the intestine.The cup cells are also apparently affected in the same way.The tca products act in the middle intestine after any administration oral or injection RH ffrench-Constant and DJ Bowen, Current Opinions in Microbiology, 1999, 12: 284-288. Purified tca products have shown oral toxicity against Manduca sixth (DL 50 of 875 ng / cm 2). }) .RH ffrench-Constant and DJ Bowen, 57 Cell. Mol. Life Sci. 828-833 (2000).

WO 99/42589 and U.S. Patent No. 6,281,413 describe the CT-type QRF of Photorhabdus luminescens . WO 00/30453 and WO 00/42855 describe the Xenorhabdus TC type proteins. WO 99/03328 and WO 99/54472 (and United States patents Nos. 6,174,860 and 6,277,823) refer to other toxins of Xenorhabdus and Photorhabdus .

Relatively recent cloning efforts in Xenorhabdus nematophilus also appear to have identified novel insecticidal toxin genes homologous with the P. luminescens tc loci. See, for example, WO 98/08388 and Morgan et al ., Applied and Environmental Microbiology and 2001, 67: 2062-69. In RH ffrench-Constant and DJ Bowen, Current Opinions in Microbiology, 1999.12: 284-288 , cosmid clones were selected directly to assess oral toxicity to other lepidoptera, Pieris brassicae . A toxic cosmid clone was sequenced orally. The sequence analysis in which the cosmid suggested that there are five different ORFs similar to the Photorhabdus tc genes; orf2 and orf5 both have some level of sequence relationship to both tcbA and tcdA, while orf1 is similar to tccB, orf3 is similar to tccC and orf4 is similar to tcaC . Importantly, numerous of these ORFs also share the supposed cleavage site documented in P. luminescens , suggesting that active toxins can also be processed proteolytically.

There are five typical Xenorhabdus TC proteins: XptA1, XptA2, XptB1, XptC1, and XptD1. XptA1 is an "autonomous" toxin. XptA2 is another Xenorhabdus TC protein that has autonomous toxin activity. See GENBANK Accession No. AJ308438 for Xenorhabdus nematophilus sequences. XptB1 and XptC1 are the potencies of Xenorhabdus that can enhance the activity of either (or both) toxins of XptA. XptD1 has some level of homology with TccB. XptC1 has some level of similarity with TcaC. Xenorhabdus XptA2 protein has some degree of similarity with TcdA protein. XptB1 has some degree of similarity with TccC.

The finding of some similarity, the loci that encode toxins in these two different bacteria is interesting in terms of the possible origins of these virulence genes. The cosmid of X. Nematophilus also appears to contain transposase-like sequences whose presence may suggest that these loci can be transferred horizontally between different strains or species of bacteria. A range of such transfer episodes can also explain the apparently different genomic organization of the tc operons in the two different bacteria. In addition, only a subset of strains of X. nematophilus and P. Luminescens seem toxic to M. sixth , suggesting either that different strains lack the tc genes or that they can carry a different complement of the tc gene. Detailed analysis of both a strain and a toxin phylogeny within, and between, these bacterial species should help clarify the probable origin of the toxin genes and how they are maintained in different populations of bacteria. RH ffrench- Constant and Bowen, Current Opinions in Microbiology, 1999, 12: 284-288.

CT proteins and genes have been described more recently from other bacteria associated with insects such as Serratia entomophila , an insect pathogen. Waterfield et al ., TRENDS in Microbiology, Vol. 9, No. 4, April 2001.

In summary, the toxin complex proteins of P. luminescens and X. Nematophilus appear to have poor homology with previously identified bacterial toxins and should provide useful alternatives to toxins derived from B. thuringiensis . Although they have similar toxic effects in the midgut of the insect to other orally active toxins, their precise mode of action remains obscure. Future work could clarify its mechanism of action.

Although some Xenorhabdus TC proteins were found to "correspond" (have a similar function and some level of sequence homology) with any of the Photorhabdus TC proteins, a given Photorhabdus protein shares only approximately 40% sequence identity with the "corresponding" Xenorhabdus protein. This is illustrated below for four "autonomous" toxins:

2

(For a more complete review, see, for example, Morgan et al ., "Sequence Analysis of Insecticidal Genes from Xenorhabdus nematophiles PMFI296", Vol. 67, Applied and Environmental Microbiology, May 2001, p. 2062-2069).

Bacteria of the genus Paenibacillus are distinguishable from other bacteria by distinctive rRNA and phenotypic characteristics (C. Ash et al . (1993), "Molecular identification of rRNA group 3 bacilli (Ash, Farrow, Wallbanks and Collins) using a probe assay PCR: Proposed for the creation of a new genus Paenibacillus ", Antonie Van Leeuwenhoek 64: 253-260). The sequence analysis of comparative 16 S rRNA showed that the genus Bacillus consisted of at least five phytic lines. Bacilli from group 3 of ribosomal RNA (from Ash, Farrow, Wallbanks, and Collins (1991), which comprise Bacillus polymyxa and closely related), is thus phylogenetically removed from Bacillus subtilis (genus type species and other aerobic, forming bacilli of endospores) that were re-classified as a new genus, Paenibacillus .

Some species in this genus will be known to be pathogenic for honey bees ( Paenibacillus Larvae ) and beetle larvae ( P. popilliae and P. lentimorbus ). Some other Paenibacillus species have been found to be associated with honey bees, but they are not pathogenic. At least 18 additional species are known in this genus, including P. thiaminolyticus; no association with insects has been known (Shida et al ., 1997; Pettersson et al., 1999). Beetles (beetles) are serious grass pests, seedbeds, and food crops throughout North America, and are of quarantine interest. See, the website of the United States Department of Agriculture, Agricultural Research Service.

P. larvae, P. popilliae, and P. lentimorbus are considered obligate insect pathogens implicated in milky beetle bacteriosis (DP Stahly et al . (1992), "The genus Bacillus: insect pathogens", p. 1697-1745, in A. Balows et al ., ed., The Procaryotes, 2nd Ed., Vol. 2, Springer-Verlag, New York, NY). These three species of Paenibacillus are characteristically slow-growing fastidious organisms that cause disease through an invasive procedure in which bacteria cross the middle intestine and proliferate to high numbers in hemolymph and other tissues. For these three species, some general indications of the involvement of protein in the pathogenicity of insects have been proposed; however, no specific role for a specific protein has been demonstrated. Stahly et al. they concluded for P. larvae that a question of the implication of a toxin is an open one, and that the precise cause of death in milky bacteriosis (of beetles) is not understood.

A beetle toxin (beetle), Cry 18, has been identified in P. popilliae and P. lentimorbus strains . Cry 18 has approximately 40% identity with Cry 2 proteins (Zhang et al., 1997; Harrison et al ., 2000). While Zhang et al . (1997) speculate that Cry 18 attacks the midgut to facilitate the entry of vegetative cells into the hemocoel, Harrison et al . They indicate that there is no direct evidence of this role and also establish that "the role, if it exists, of the protein of paraspores in milky bacteriosis is not known" J. Zhang et al . (1997), "Cloning and Analysis of the First cry Gene from Bacillus popilliae ", J. Bacteriol. 179: 4336-4341; H. Harrison et al . (2000), " Paenibacillus Associated with Milky Disease in Central and South American Scarabs", J. Invertebr. Pathol 76 (3): 169-175.

Stahly et al ., Zhang et al ., And Harrison et al . all point to the contrast in evidence for the role of B crystalline proteins. Thuringiensis in insect intoxication (where the high frequency of insect symptoms can be explained by specific crystalline protein proteins), against the case of Paenibacillus and milky bacteriosis (where there is no such link for the effects of a specific toxin) .

Thus, while some Paenibacillus species are known to be pathogenic for certain beetles and some associated to certain beetles and some associated to honey bees, no strain of Paenibacillus that was toxic to lepidoptera was known until now. Similarly, CT proteins and toxic Cry proteins for lepidoptera have never been reported in Paenibacillus .

Brief summary of the invention

This is the first known description of Paenibacillus protein toxins that have activity against lepidopteran pests. Some Paenibacillus species are known to be insecticides, but have no activity against weevils / beetles / beetles. There have been no known reviews of a species or strain of Paenibacillus that has toxicity to lepidoptera. Thus, the present invention relates generally to the selection of Paenibacillus spp. For proteins that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41, and their use.

More specifically, the present invention initially came from a discovery of a novel strain of Paenibacillus mentioned herein as DAS1529. This was a surprising discovery for a variety of reasons. This strain produces a unique toxic protein for lepidoptera. This strain, as well as DB482, produces unique toxin complex type CT proteins (which have some similarity with Xenorhabdus / Photorhabdus CTs).

This is the first known review of Paenibacillus that has TC-type proteins. Thus, the present invention relates to methods of selecting Paenibacillus spp. For genes and proteins of the TC type as before. Paenibacillus Tc proteins of the present invention are shown herein that are useful for increasing or enhancing the activity of an "autonomous" toxin protein of Xenorhabdus , for example. The TC-type genes identified in this document were not known until now that they existed in the genus Paenibacillus . The discovery broadens the scope of organisms (bacterial genus in which the TC type genes have been found. Thus, the present invention generally refers to TC type proteins as before they can be obtained from Paenibacillus species , to procedures for selecting Paenibacillus species for such proteins, and the like An example is Paenibacillus apairius , which is also found to produce TC-like proteins.

While the subject CT type proteins have some sequence connection, and features in common with the Xenorhabdus and Photorabdus type TC proteins , the sequences of the subject CT type proteins are very different from the previously known CT proteins. Thus, the subject application provides new classes of TC-type proteins and genes that encode these proteins, which can be obtained from bacteria in the genus Paenibacillus,.

Another striking feature of strain DAS 1529 is that it produces a single Cry type B protein. t . which is toxic to lepidoptera. The subject Cry toxin is compressed / short and appears to lack a typical protoxin portion in its wild-type state.

This strain is the first known example of a natural bacterium that produces both a Cry type toxin and TC type proteins. It is also surprising that this is the first known example of a cry toxin gene that is closely associated with (in genetic proximity to) the TC protein genes. These pioneering observations have broad implications and thus allow those skilled in the art to select appropriate species of bacteria for these types of unique operons and for these types of additional operon components.
known.

A further description of the present invention comes from the surprising discovery that strain DAS 1529 also produces a soluble insect toxin that was found to be very similar to a thiaminase. It was surprising that Paenibacillus thiaminase protein was found to have insecticidal activity. While this protein was known, it was not expected in any way in the art that this enzyme had shown toxin-like activity against insect / insect pests.

Other objects, advantages, and characteristics of the Present invention will be apparent to those skilled in the art. They have the benefit of the subject description.

Figure 4 shows test results of purified thiaminase from DAS 1529 in CEW.

Figure 5 shows ORF3-ORF6 in pEt101D.

Figure 6 shows Cry 1529 (ORF 7) against the tobacco yolk worm (TBW).

Figure 7 shows a comparison / alignment of SEQ ID NO: 9 to SEQ ID NO: 5 (tcaB2 to tcaB1); The parentheses show the connection of ORF2.

Figure 8 shows a phylogenetic tree of ORF7 from DAS1529 ( Cry 1529) compared to other Cry proteins.

Figures 9 and 10 show the results of trypsin digestion of wild-type and modified Cry 1529 proteins.

Figures 11A and 11B show the alignments of sequences for the tcaA primer design.

Figures 12A-D show the sequence alignments for tcaB primer design.

Figures 13A and 13B show the alignments of sequences for the tcaC primer design.

Figures 14A and 14B show the alignments of sequences for the tccC primer design.

Brief description of the sequences

SEQ ID NO: 1 is the acid sequence nucleus of the entire SB12 insert.

SEQ ID NO: 2 is the ORF1 nucleic acid sequence, which encodes a tcaA type protein ( tcaA1 gene, strain IDAS 1529 of Paenibacillus from the source organism designation of the tcaA1-1529 gene).

SEQ ID NO: 3 is the amino acid sequence encoded by ORF1.

SEQ ID NO: 4 is the ORF2 nucleic acid sequence, with an IS element removed, that encodes a tcaB-like protein ( tcaB1 gene, Paenibacillus strain IDAS 1529 from the source organism, designation of the tcaB1-1529 gene).

SEQ ID NO: 5 is the amino acid sequence encoded by ORF2.

SEQ ID NO: 6 is the nucleic acid sequence of ORF3 encoding a protein of TCAA type (tcaA2 gene, strain IDAS 1529 of the source organism Paenibacillus, designation tcaA2-1529 gene).

SEQ ID NO: 7 is the amino acid sequence encoded by ORF3.

SEQ ID NO: 8 is the ORF4 nucleic acid sequence, which encodes a tcaB type protein ( tcaB2 gene, strain Paenibacillus IDEAS 1529 from the source organism, designation of the tcaB2-1529 gene).

SEQ ID NO: 9 is the amino acid sequence encoded by ORF4.

SEQ ID NO: 10 is the nucleic acid sequence of ORF5 encoding a protein type TCAC (TCAC gene, strain IDAS 1529 of the source organism Paenibacillus, designation TCAC-1529 gene).

SEQ ID NO: 11 is the amino acid sequence encoded by ORF5.

SEQ ID NO: 12 is the acid sequence ORF6 nucleic, which encodes a tccC type protein.

SEQ ID NO: 13 is the amino acid sequence encoded by ORF6.

SEQ ID NO: 14 is the nucleic acid sequence of ORF7, which encodes a Cry type protein.

SEQ ID NO: 15 is the amino acid sequence encoded by ORF7.

SEQ ID NO: 16 is the acid sequence partial nucleus of the 16S rDNA of DAS 1529 used for placement taxonomic

SEQ ID NO: 17 is the amino acid sequence N-terminal for purified toxin fraction of broth from DAS1529.

SEQ ID NO: 18 is the amino acid sequence of thiaminase I from Bacillus thiaminolyticus (Campobasso et al ., J. Biochem. 37 (45): 15981-15989 (1998)).

SEQ ID NO: 19 is an alternative amino acid sequence encoded by the ORF6 protein ( tccC gene , strain IDAS 1529 of Paenibacillus from the source organism, designation of the tccC-1529 gene).

SEQ ID NO: 20 is the xptC1 gene , Xwide Xenorhabdus strain of the source organism, designation of the xptC1-Xwi gene.

SEQ ID NO: 21 is the xptB1 gene , Xwide Xenorhabdus strain of the source organism, designation of the xptB1-Xwi gene.

SEQ ID NO: 22 is the SB101 primer.

SEQ ID NO: 23 is the SB102 primer.

SEQ ID NO: 24 is the SB103 primer.

SEQ ID NO: 25 is the SB104 primer.

SEQ ID NO: 26 is the SB105 primer.

SEQ ID NO: 27 is the SB106 primer.

SEQ ID NO: 28 is the SB212 primer.

SEQ ID NO: 29 is the SB213 primer.

SEQ ID NO: 30 is the SB215 primer.

SEQ ID NO: 31 is the SB217 primer.

SEQ ID NO: 32 is a nucleotide sequence of a tcaA type gene of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 33 is an amino acid sequence of a TcaA type protein of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 34 is a nucleotide sequence of a tcaB type gene of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 35 is a nucleotide sequence of a tcaB type gene of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 36 is an amino acid sequence of a TcaB type protein from strain DB482 of Paenibacillus apairius .

SEQ ID NO: 37 is an amino acid sequence of a TcaB type protein of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 38 is a nucleotide sequence of a tcaC type gene of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 39 is an amino acid sequence of a TcaC type protein from strain DB482 of Paenibacillus apairius .

SEQ ID NO: 40 is a nucleotide sequence of a tccC type gene of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 41 is an amino acid sequence of a TccC type protein of strain DB482 of Paenibacillus apairius .

SEQ ID NO: 42 is the tcdB1 gene , strain W14 of Photorhabdus of the source organism, designation of the tcdB1-W14 gene.

SEQ ID NO: 43 is the tcdB2 gene , strain W14 of Photorhabdus of the source organism, designation of the tcdB2-W14 gene.

SEQ ID NO: 44 is the tccC1 gene , strain W14 of Photorhabdus of the source organism, designation of the tccC1-W14 gene.

SEQ ID NO: 45 is the tccC2 gene , strain W14 of Photorhabdus of the source organism, designation of the tccC2-W14 gene.

SEQ ID NO: 46 is the tccC3 gene , Photorhabdus strain W14 of the source organism, designation of the tccC3-W14 gene.

SEQ ID NO: 47 is the tccC4 gene , strain W14 of Photorhabdus of the source organism, designation of the tccC4-W14 gene .

SEQ ID NO: 48 is the tccC5 gene , strain W14 of Photorhabdus of the source organism, designation of the tccC5-W14 gene .

SEQ ID NO: 49 is the amino acid sequence of the XptA2 TC protein of Xenorhabdus nematophilus Xwi.

Detailed description of the invention

The present invention provides unique biological alternatives for pest control. More specifically, the present invention provides novel sources of proteins that have activity of toxins against insects, preferably lepidoptera, and other similar pests. The invention also relates to novel sources of novel polynucleotides that can be used to encode such toxins, and to preparation procedures and procedures for using the corresponding toxins and nucleic acid sequences to control insects and other plant pests. The present invention relates to the need for novel insect control agents. The present invention to novel pesticide proteins that can be obtained from Paenibacillus bacteria.

The present invention initially comes from a discovery of a novel strain of Paenibacillus. This strain is referred to herein as DAS1529. To demonstrate the broad implications of this discovery, the discovery of another strain of Paenibacillus is also exemplified. These strains have been deposited in the Patent Crops Collection of the Agricultural Research Service (NRRL) in 1815 North University Street Peoria, Ill. 61604 United States. The strains deposited and the corresponding deposit dates are as follows:

2000

These crops have been deposited for purposes of this patent application and were deposited in the conditions that ensure that access to crops is available during the pending status of this patent application to that determined by the Commissioner of Patents and Trademarks which is also called 37 CFR 1.14 and 35 U.S.C. 122. These deposits will be available when required by patent laws foreigners in countries where the application counterparts subject, or their descendants present themselves. However it is due understand that the availability of a deposit does not constitute a license to practice the present invention in the repeal of of patent rights granted by the action governmental.

In addition, subject culture deposits are performed in accordance with the provisions of the Budapest Treaty for the deposit of Microorganisms, that is, they will be stored with all the necessary care to keep them viable and not contaminated for a period of at least five years after the most recent request for the supply of a sample of deposit, and in any case for a period of at least (30) years after the deposit date or for the executive life of Any patent that can publish the description of the crop. He depositor acknowledges the duty to replace the deposit the deposited must be unable to provide a sample when require, due to the condition of the deposit. All restrictions on the availability of deposits to the public of subject crop will be irrevocably removed upon grant of a patent that describes them.

The discovery of the subject strain DAS1529 was surprisingly due to a variety of reasons. The strain produces a unique Cry protein toxic to lepidoptera. This strain, as well as DB482, also produces toxin complex type (CT) proteins (which have some similarity to Xenorhabdus / Photorhabdus CTs).

This is the first known description of a Paenibacillus protein toxin that has activity against a lepidopteran pest. This was a surprising discovery. Some species of Paenibacillus were known to have insecticidal activity against weevils / beetles / beetles. There have been no known reviews of a species or strain of Paenibacillus that has toxicity to lepidoptera. Thus, the present invention generally describes Paenibacillus species that have activity against lepidoptera, and selection of Paenibacillus cultures, their proteins, and clone libraries thereof, for activity against lepidoptera, and / or for genes that encode "lep toxins", and more particularly, for Cry proteins toxic to lepidoptera.

This is also the first known review of Paenibacillus that has TC type proteins. Thus, the present invention relates to methods of selecting Paenibacillus spp. For genes and proteins of the TC type that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41. It was very surprising to find that strains DAS 1529 and DB482 have TC-type operons. and produce these TC proteins (which have some level of similarity with the Xenorhabdus and Photorhabdus TC proteins). The TC proteins and genes identified in this document were not known until now that exist in the genus Paenibacillus . This discovery extends the scope of organisms (bacterial genera) in which the genes of the TC proteins have been found. Thus, the present invention generally refers to TC proteins that can be obtained from Paenibacillus species that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41, and to procedures for selecting Paenibacillus species for such proteins having an amino acid sequence selected from the group consisting of SEQ ID No 13, and SEOQ ID No. 41. An example of a species of Paenibacillus found using the methods of the present invention is strain DB482 of Paenibacillus apairius . This strain of P. apairius also produces unique TC-type proteins that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID
No. 41

Although the subject CT proteins have some characteristics in common with the Xenorhabdus and Photorabdus TC proteins, the subject TC proteins are unique and different from the previously known TC proteins. In this way, the subject application provides new classes of proteins and genes of the TC type that encode these proteins that can be obtained from bacteria in the genus Paenibacillus .

Dry1529 Cry toxin is a type B Cry protein toxin. t .. Another strain of Paenibacillus, a strain with activity against weevils, was known to produce a toxic Cry protein for beetles. That was a Cry 18 protein, which was more related to Cry 2 proteins (but only about 40% identity). The Cry protein exemplified herein shows only a low level of identity and sequence similarity to previously known Cry proteins. With that indicated, of all the Cry proteins of Bt Cry , the subject Cry protein shares most of the similarity with the Cry 1 proteins. A surprising aspect of the subject Cry protein is that it is very short, that is even shorter than Cry 1Fa core toxin. The subject Cry protein has a region 5 of block that can be identified in or near its C This toxin in its wild type state is not part of the protoxin, which is typically in the Cry Cry toxin 1. toxins the subject is surprisingly compressed. This description is also significant for the investigation of additional cry genes of Bacillus thuringiensis ( Bt ). As will be apparent to those skilled in the art who have the benefit of the subject description, other bacteria, such as B. t . and other Bacillus spp. They can be selected for similar toxins and toxin genes.

The strain DAS1529 is the first known example of a natural bacterium that produces both Cry type toxin and TC type proteins. Also surprisingly, this is the first known example of a cry toxin gene that is closely associated with (in genetic proximity c) CT protein genes. These pioneering observations thus allow those skilled in the art to select appropriate species of bacteria for these types of unique operons and for these types of additional components of known operons. This is an interesting example of a wild-type strain that has an operon. TC type with multiple CT protein genes of the same general type (i.e., in this case, two tcaA type and two tcaB type genes). These surprising discoveries have broad implications for the discovery of additional genes.

An additional description derives from the surprising discovery that Paenibacillus thiaminase protein has insecticidal activity. Although this protein was known, it was not expected in any way in the art that this enzyme would show toxin-like activity against insect / insect pests.

Paenibacillus TC proteins

More specifically in relation to the exemplified TC proteins, the following TC proteins of strain DAS 1529 have been fully characterized herein: two TcaA type proteins (TcaA1 and TcaA2), two TcaB type proteins (TcaB1 and TcaB2), a TcaC protein, and a TccC type protein. The TcaA1 and TcaA2 proteins are largely similar to each other at the sequence level, and the tcaB1 and tcaB2 proteins are similar to each other at the sequence level. The TC-type proteins that can be obtained from Paenibacillus apairius are also exemplified herein.

The TC proteins of the present invention having an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 can be used as other TC proteins. This would be readily apparent to those skilled in the art who have the benefit of the subject description when viewed in light of what is known in the art. See, for example, the previous background section, which describes RH ffrench-Constant and Bowen (2000) and U.S. Patent No. 6,048,838. For example, it was known that Photorhabdus Tca toxin complex is highly toxic to Manduca sixth .

Although the exact molecular interactions of the TC proteins with each other, and their mechanism (s) of action, are not currently understood, some TC proteins were known to have "autonomous" insecticidal activity, and other TC proteins were known to potentiate the activity of the autonomous toxins produced by the same given organism. For example, it was known that TcdA protein was active against Manduca sixth . TcaC and TccC, can be used together to enhance the activity of TcdA. TcdB can be used (instead of TcaC) with TccC as an enhancer. TcbA is another Photorhabdus TC protein with autonomous toxic activity. TcaC (or TcdB) in conjunction with TccC can also be used to increase / enhance the toxic activity of TcbA.

The Photorhabdus CT proteins and "corresponding" CT proteins / genes of Paenibacillus are summarized below.

3

As indicated above, TccA has some level of homology with the N end of TcdA, and TccB has some level of homology with the C end of TcdA. In addition, TcdA is of approximately 280 kDa, and TccA together with TccB are of approximately the same size, if combined, than TcdA. Further, TcaA has some level of homology with TccA and similarly with the N end of TcdA. Even in addition, TcaB has some level of homology with TccB and similarly with the N-terminus of TcdA. TccA and TcaA are of similar size, than TccB and TcaB.

Although some Xenorhabdus TC proteins were found to "correspond" with some of the Photorhabdus TC proteins, the "corresponding" proteins share only approximately 40% (approximately) sequence identity with each other. Paenibacillus CT subject CT proteins have approximately the same degree of sequence connection (~ 40% identity) with the previous CT proteins.

As described in more detail below, one or more toxins of the present invention can be used in combination with each other and / or with other toxins (ie, the Photorhabdus Tca complex was known to be active against Manduca sixth ; various " combinations of " Photorhabdus TC proteins, for example, can be used together to enhance the activity of other, autonomous Photorhabdus toxins; the use of Photorhabdus toxins" with " B toxins. t ., for example, has been proposed for resistance management). In addition, the Paenibacillus TC proteins of the present invention having an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 are shown herein that are useful for increasing or enhancing the activity of an "autonomous" Xenorhabdus toxin protein, for example. Provisional application No. 60 / 441,723 (Timothy D. Hey et al .), Entitled "Mixing and Matching of TC Proteins for Pest Control", refers to the surprising discovery that a TC protein derived from an organism of such a genus As Photorhabdus can be used interchangeably with a "corresponding" TC protein derived from an organism of another gender, In addition, the surprising data along with these lines are presented below that further illustrate the usefulness of the Paenibacillus CT proteins of the present invention. which have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 . One reason that these results might be surprising is that there is only? 40% sequence identity between the "corresponding" TC proteins of Xhenorhabdus, Photorhabdus , and subject Paenibacillus .

Proteins and toxins . The present invention easily administered functional proteins having an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 . The present invention also provides a method for distributing insecticidal toxins that are functionally active and effective against many orders of insects, preferably lepidopteran insects. By "functional activity" (or "active against") means herein that protein toxins having an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 function as control agents. of orally active insects (alone or in combination with other proteins), that the proteins have a toxic effect (alone or in combination with other proteins), or are capable of disrupting or stopping growth and / or feeding of insects that may or they cannot cause insect death. When an insect is contacted with an effective amount of a "toxin" of the present invention distributed the expression of transgenic plants, formulated protein composition (s), sprayable protein composition (s), a bait matrix or other distribution system, the results are typically insect death, growth inhibition and / or prevention of insects from feeding on the source (preferably a transgenic plant) that makes toxins available for insects The functional proteins of the present invention can also enhance or improve the activity of the other protein toxins. The terms "toxic", "toxicity", and "toxic activity" as used herein means that the subject "toxins" have "functional activity" as defined herein.
document.

Full lethality is preferred for feeding but it is not required to achieve functional activity. If a insect avoids toxin or stops feeding, that avoidance It will be useful in some applications, even if the effects are sublethal or if lethality is delayed or indirect. For example, if insect-resistant transgenic plants are desired, the renunciation of insects to feed on plants is so useful as the lethal toxicity for insects because the latter objective is to avoid damage to plants induced by insects.

There are many other ways in which Toxins can be incorporated into an insect diet. For example, it is possible to adulterate the food source of the larvae with the toxin protein spraying the food with a solution of protein, as described herein. How alternatively, the modified protein can be modified by genetic engineering in another non-harmful bacteria, which then it can be grown in culture, and either applied to the power supply or allow it to reside on the ground in an area in which the eradication of the insect will be desired. Also, the protein can be engineered directly in A source of insect food. For example, the source of main food for many insect larvae is material vegetable. Therefore genes that encode toxins can be transfer to the plant material so that said material of plant express the toxin of interest.

The transfer of functional activity to plant or bacterial systems typically requires sequences of nucleic acid, which encode the amino acid sequences for toxins, integrated into an appropriate protein expression vector for the host in which the vector will reside. A way to get a nucleic acid sequence that encodes a protein with Functional activity is to isolate the native genetic material from of the bacterial species that produce the toxins, using the information deduced from the amino acid sequence of the toxin, as described in this document. The native sequences are they can optimize for expression in plants, for example, as describe in more detail below. The optimized polynucleotide It can also be designed based on the protein sequence.

The present invention provides new classes of toxins that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 that have advantageous pesticidal activities. One way to characterize three classes of toxins and the polynucleotides that encode them is by defining a polynucleotide by its ability to hybridize, in a range of specified conditions, with an exemplified nucleotide sequence (its complement and / or a probe or probes derived from any strand) and / or by its ability to amplify by PCR using primers derived from the exemplified sequences.

There are numerous methods for obtaining the pesticidal toxins of the present invention that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 . For example, antibodies to the pesticidal toxins described and claimed herein can be used to identify and isolate other toxins from a mixture of proteins. Specifically, antibodies can be induced for the parts of the toxins that are the most constant and the most different from other toxins. These antibodies can then be used to specifically identify equivalent toxins with the characteristic activity by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), or western blotting. Antibodies to the toxins described herein, or to equivalent toxins, or to fragments of these toxins, can be easily prepared using conventional procedures. The toxins of the present invention can be obtained from a variety of source / source microorganisms.

Those skilled in the art would readily recognize that the toxins (and genes) of the present invention can be obtained from a variety of sources. A "de" or "toxin that can be obtained from" the subject isolation DAS 1529 and / or the P. apiarius isolation means that the toxin (or a similar toxin) can be obtained from this isolation or some other source, such as another bacterial strain or a transgenic plant. For example, those skilled in the art will readily recognize that, given the description of a bacterial gene and toxin, a plant can be engineered to produce the toxin. Antibody preparations, nucleic acid probes (DNA and RNA), and the like can be prepared using the polynucleotide and / or amino acid sequences described herein and used to select and recover other toxin genes from other sources.
(natural)

Polynucleotides and probes . The present invention further describes nucleotide sequences encoding the toxins of the present invention that have an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 . The present invention further provides methods for identifying and characterizing genes encoding pesticidal toxins. In one embodiment, the present invention describes unique nucleotide sequences that are useful as hybridization probes and / or primers for PCR techniques. The primers produce characteristic gene fragments that can be used in the identification, characterization and / or isolation of genes from specific toxins. Nucleotide sequences as described encode toxins that are different from the toxins described above.

The polynucleotides as described can be used to complete "genes" that encode proteins or peptides in a desired host cell. For example, as those skilled in the art will readily recognize, subject polynucleotides can be appropriately placed under the control of a promoter, in a host of interest, as is readily known in the art.

As those skilled in the art know, DNA typically exists, in a double stranded form. In this arrangement, one strand is complementary to the other strand and vice versa. As DNA is replicated in a plant (for example), additional complementary strands of AD are produced. The custom "coding strand" is used in the art to refer to the strand that binds with the non-coding strand. The mRNA is transcribed from the "non-coding" strand of DNA. The "positive sense" or "coding" strand has a series of codons (a codon is three nucleotides that can be read as a unit of three residues to specify a particular amino acid) that can be read as an open reading frame (ORF ) to form a protein or peptide of interest. In order to express a protein in vivo , a strand of DNA is typically transcribed into a complementary strand of mRNA that is used as the protein template. Thus, the present invention describes the use of the exemplified polynucleotides shown in the linked sequence listing and / or equivalents that include the complementary strands. RNA and PNA (peptide nucleic acids) that are functionally equivalent to DNA are described
exemplified.

Bacterial isolates can be grown under conditions that result in a high multiplication of of the microbe. After treating the microbe to provide acid single-chain genomic nucleic, And can be put into contact with the primers of the invention and undergo PCR amplification. The characteristic gene fragments that encode toxin will be amplified by the procedure, thus identifying the presence of the gene (s) encoding toxin

In addition , genes and isolates identified using the nucleotide procedures and sequences described herein are described. The genes identified in this way encode active toxins against pests.

Toxins having an amino acid sequence selected from the group consisting of SEQ ID No. 13, and SEQ ID No. 41 and genes of the present invention can be identified and obtained using oligonucleotide probes, for example. These probes are detectable nucleotide sequences that can be detected under an appropriate tag or can be made fluorescent in an interesting manner as described in International Application No. WO 93/16094. The probes (and polynucleotides of the present invention) can be DNA, RNA, or PNA. In addition to the adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U; for RNA molecules), synthetic probes (and polynucleotides) of the present invention may also have inosine (a Neutral base capable of mating with the four bases; sometimes used instead of a mixture of the four bases in synthetic probes). Thus, when a synthetic, degenerate oligonucleotide is mentioned herein, and "n" is used <generically, "n" may be G, A, T, C, or inosine. The ambiguity codes as used herein are in accordance with conventional IUPAC nomenclature conventions such as that of submitting the subject application (for example, R means A or G, Y means C or T, etc.).

As is well known in the art, if a probe molecule hybridizes with a sample of nucleic acid, it can reasonably assume that the probe and sample have homology / similarity / substantial identity. Preferably the Hybridization of the polynucleotide is first carried out by washed in conditions of low, moderate, or high stringency by techniques well known in the art, as described in,  for example, Keller, G.H., M.M. Manak (1987) DNA Probes, Stockton Press, New York, NY, p. 169 - 170. For example, as stated in that document, low stringency conditions can be achieved by washing with 2 x SSC Citrate Standard saline solution) / 0.1% of SDS (Sodium Dodecyl Sulfate) for 15 minutes at temperature ambient. Two washes are typically performed. Then you can achieve greater rigor by reducing the salt concentration and / or raising the temperature For example, to the wash described previously you can follow two washes with 0.1 x SSC / 0.1% SDS for 15 minutes each at room temperature followed by subsequent washes with 0.1 x SSC / 0.1% SDS for 30 minutes each one at 55 ° C. These temperatures can be used with other protocols of hybridization and washing established in this document and as those skilled in the art will know (SSPE can be used as salt instead of SSC, for example). 2 x SSC / 0.1% SDS is can be prepared by adding 50 ml of 20 x SSC and 5 ml of 10% SDS to 445 ml of water. 20 x SSC can be prepared using the combination of NaCl (175.3 g / 0.150 M), sodium citrate (88.2 g / 0.015 M), and water up to 1 liter, followed by pH adjustment to 7.0 with 10 N NaOH 10% SDS can be prepared by dissolving 10 g of SDS in 50 ml of sterilized water in an autoclave, diluting up to 100 ml, and taking aliquots.

Probe detection provides a means to determine in a known way if hybridization has been maintained. Such probe analysis provides a rapid procedure to identify the genes encoding toxin of the present invention. Nucleotide segments that are used as probes as described in accordance with the invention can be synthesized using a DNA synthesizer and conventional procedures. These nucleotide sequences can also be used as PCR primers to amplify genes of the invention.
Present.

The probes for use according to the invention present can be derived from a variety of sources, such as any gene mentioned or suggested in this document. By example, all or part of any of the genes (encoders and / or non-coding or its complementary strands) can be used in according to the present invention: tcaA, tcaB, tcaC, tcbA, tccA, tccB, tccC, tcdA, tcdB, xptA1, xptD1, xptB1, xptc1, xptA2, sepA, sepB, and sepC. Unless otherwise specified, the reference to a "tccC" gene, for example, includes all alleles specific (such as tccC1 and tccC2) of this type of gene. The same It is true for all other genes (for example, tcdB2, tccC3, and the alleles mentioned in Table 17).

The hybridization characteristics of a molecule can be used to define the polynucleotides of the present invention. Thus, the present invention describes polynucleotides (and / or their supplements, preferably their complete complements) that hybridize with a polynucleotide (or a oligonucleotide or primer) exemplified or suggested herein document.

As "stringent" conditions for hybridization are used herein, it refers to conditions that achieve the same, or approximately, the same degree of hybridization specificity as the conditions employed by current applicants. Specifically, hybridization of immobilized DNA on Southern blots with 32P-labeled gene-specific probes was performed by conventional procedures ( see, for example, Maniatis, T., EF Fritsch, J. Sambrook [1982] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). In general, hybridization and subsequent washing can be carried out under conditions that allow detection of target sequences. For double stranded DNA gene probes, hybridization was carried out overnight 20-25 ° C below the melting temperature (Tm) of the DNA hybrid in 6 x SSPE, 5x Denhardt's solution, 0 , 1% SDS, 0.1 mg / ml denatured DNA. The melting temperature is described by the following formula (Beltz, GA, KA Jacobs, TH Eickbush, PT Cherbas, and FC Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave [eds.] Academic Press, New York 100: 266-285):

Tm = 81.5 ^ o C \ + \ 16.6 \ Log [Na +] \ + \ 0.41 (% G + C) - 0.61 (% formamide) - 600 / length \ of duplex \ in \ pairs \ of \ bases.

Washings are typically carried out as follow:

(1) Twice at room temperature for 15 minutes in 1 x SSPE, 0.1% SDS (low stringency wash).

(2) Once at Tm - 20ºC for 15 minutes in 0.2 x SSPE, 0.1% SDS (wash with moderate stringency).

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For oligonucleotide probes, the Hybridization was carried out overnight at 10-20 ° C by below the melting temperature (Tm) of the hybrid at 6 x SSPE, 5 x Denhardt solution, 0.1% SDS, 0.1 mg / ml DNA denatured. Tm for oligonucleotide probes was determined by the following formula:

Tm (° C) = 2 \ (number \ of \ pairs \ of \ bases \ T / A) + 4 \ (number \ of \ pairs \ of \ bases\ G / C)

(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura, and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes, D.D. Brown [ed.], Academic Press, New York, 23: 683 - 693).

The washings were typically carried out as follow:

(1) Twice at room temperature for 15 1x SSPE minutes, 0.1% SDS (low stringency wash).

(2) Once at hybridization temperature for 15 minutes in 1 x SSPE, 0.1% SDS (stringency wash moderate).

In general, salt and / or temperature can be alter to change the rigor. With a DNA fragment> 70 or approximately bases in length, the following conditions:

Low:

1 or 2 x SSPE, room temperature

Low:

1 or 2 x SSPE, 42 ° C

Moderate:

0.2 x or 1 x SSPE, 65 ° C

Tall:

0.1 x SSPE, 65 ° C.

Duplex formation and stability depend on the substantial complementarity between the two strands of a hybrid and two strands of a hybrid, and as indicated above, a certain degree of disagreement can be tolerated. Therefore, the probe sequences as described , include mutations (both individual and multiple), deletions, insertions of the described sequences, and combinations thereof, in which said mutations, insertions and deletions allow the formation of stable hybrids with the target polynucleotide of interest. Mutations, insertions, and deletions can occur in a given polynucleotide sequence in many ways, and these procedures are known to those skilled in the art. Other procedures will be known in the
future.

PCR technology : Polymerase chain reaction (PCR) is a repetitive, enzymatic, barley synthesis of a nucleic acid sequence. This procedure is well known and is commonly used by those skilled in the art (see Mullis, U.S. Patent Nos. 4,683,195, 4,683,202. And 4,800,159; Saiki, Randall K., Stephen Scharf, Fred Faloona, Kary B. Mullis, Glenn T. Horn, Henry A. Erlich, Norman Arnheim "Enzymatic Amplification of β-Globin Genomic Sequences and Restriction Site Analysis for Diagnosis of Sickle Cell Anemia", Science 230: 1350-1354). PCR reactions are based on the enzymatic amplification of a DNA fragment of interest that is flanked by two oligonucleotide primers that hybridize with opposite strands of the target sequence. The primers are oriented with the 3 N ends pointing towards another. Repeated cycles of heat denaturation of the template, hybridization of the primers with their complementary sequences, and extension of the primers hybridized with an ADn polymerase results in the amplification of the segment defined by the 5 N ends of the PCR primers. The extension product of each primer can serve as a template for the other primer, so each cycle essentially doubles the amount of DNA fragment produced in the previous cycle. This results in the exponential accumulation of specific target fragments, up to several million times in a few hours. By using a thermostable DNA polymerase such as Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus , the amplification procedure can be fully automated. Other enzymes that can be used are known to those skilled in the art.

DNA sequences as described in the present invention can be used as primers for PCR amplification. In the realization of PCR amplification, a certain degree of mismatch between primer and template can be tolerated. Therefore, mutations, deletions, and insertions (especially nucleotide additions to the 5 N end) of the exemplified primers fall within the scope of the present invention. Mutations, insertions and deletions can be produced in a given primer by methods known to those skilled in the art.

Modifications of genes and toxins . The genes and toxins useful in accordance with the present invention include not only specifically exemplified full length sequences, but also portions, segments and / or fragments (including internal and / or terminal deletions compared to full length molecules) of these sequences, variants, mutants, chimerics, and their fusions. For example, toxins of the present invention can be used in the form of chimeric toxins produced by the combination of portions of two or more toxins / proteins.

The proteins of the present invention may have substituted amino acids as long as they can maintain the characteristic pesticidal / functional activity of the proteins exemplified specifically herein. "Variant" genes have nucleotide sequences that encode the same toxins or equivalent toxins that have equivalent pesticidal activity with an exemplified protein. The terms "variant proteins" and "equivalent toxins" refer to toxins that have the same or essentially the same biological / functional activity against target pests and equivalent sequences as exemplified toxins. As used herein, reference to an "equivalent" sequence refers to sequences that have substitutions, deletions, additions, or insertions of amino acids, which enhance or not adversely improve pesticidal activity. Fragments that retain pesticidal activity are also included in this definition. Fragments and other equivalents that retain the same or similar function, or "toxin activity", of a corresponding fragment of a toxin are within the scope of the present invention. Changes may be made, such as amino acid substitutions or additions for a variety of purposes, such as increasing (or decreasing) the protease stability of the protein (without materially / substantially decreasing the functional activity of the protein).
toxin).

The equivalent toxins and / or genes encoding these equivalent toxins can be obtained / derived from wild-type or recombinant bacteria and / or other wild-type or recombinant microorganisms using the teachings provided herein. Other Paenibacillus species, for example, can be used as source isolates.

Gene variations can be easily constructed using conventional techniques to prepare point mutations, for example. In addition, U.S. Patent No. 5,605,793, for example, describes methods for generating additional molecular diversity by using DNA reassembly after random fragmentation. Variant genes can be used to produce variant proteins. Using these "gene recomposition" techniques, equivalent genes and proteins comprising any 5, 10, or 20 contiguous moieties (amino acids or nucleotide) of any sequence exemplified herein can be constructed. As those skilled in the art know, the gene recomposition technique can be adjusted to have, for example, 1,2,3,4,5,6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, or 500 or more contiguous moieties (amino acids or nucleotide), corresponding to a segment (of the same size) in any of the exemplified sequences (or the complements (total complements) thereof). Similarly sized segments, especially those for conserved regions, can also be used as probes and / or
primers

Fragments of the full length genes can be prepared using commercially available exonucleases or endonucleases according to conventional procedures. For example, enzymes such as Bal 31 or site-directed mutagenesis can be used to systematically cut the ends of these genes. Also, genes encoding active fragments can be obtained using a variety of restriction enzymes. Proteases can be used to directly obtain active fragments of these
toxins

It is within the scope of the invention as described herein that the toxins may be truncated and still maintain functional activity. By "truncated toxin" is meant that a portion of a toxin protein can be cleaved and still maintain functional activity after excision. Excision can be achieved by proteases inside or outside the insect's intestine. In addition, effectively cleaved proteins can be produced using molecular biology techniques in which the DNA bases encoding said toxin are removed either by restriction endonuclease digestion or other techniques available to those skilled in the art. After truncation, said proteins can be expressed in heterologous systems such as E. coli , baculovirus, viral systems based on plants, yeasts and the like and then placed in insect assays as described herein to determine activity. It is well known in the art that truncated toxins can be produced successfully so as to retain functional activity while having less than the entire, full length sequence. It is well known in the art that Bt toxins toxins can be used truncated (core toxin). See, for example, Adang et al ., Gene 36: 289-300 (1985), "Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus thuringiensis subsp kurstaki HD-73 and their toxicity to Manduca sixth ". There are other examples of truncated proteins that retain insecticidal activity, including juvenile esterase hormone (US Patent No. 5,674,485 to the Regenets of the University of California). As used herein, the term "toxin" also means that it includes functionally active truncations.

Certain toxins of the present invention have been specifically exemplified herein by SEQ ID No. 13, and 41 . It should be readily apparent that the present invention comprises variant or equivalent toxins (and nucleotide sequences encoding equivalent toxins) that have the same or similar pesticidal activity of the exemplified toxin. The equivalent toxins will have similarity (and / or homology) of amino acids with an exemplified toxin. The amino acid identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. The preferred polynucleotides and proteins of the present invention can also be defined in terms of more particular identity and / or similarity intervals. For example, the identity and / or similarity may be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 , 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 , 94, 95, 96, 97, 98, or 99% when compared to a sequence exemplified herein. Unless otherwise specified, as used herein, percent identity and / or sequence similarity of two nucleic acids is determined using the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87: 2264-2268, modified in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al . (1990), J. Mol. Biol. 215: 402-410. BLAST nucleotide investigations are performed with the NBLAST program, score = 100, word length = 12. To obtain the alignment of gaps for comparison purposes, use, Gapped BLAST as described in Altschul et al . (1997), Nucl. Acids Res. 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) are used. See the NCBI / NIH website. Scores can also be calculated using the procedures and algorithms of Crickmore et al . As described in the background section,
previously.

The homology / similarity / identity will be the older in the critical regions of the toxin that are responsible of biological activity or are involved in determining the three-dimensional configuration that is ultimately responsible of biological activity. In this regard, certain substitutions of Amino acids are acceptable and can be expected to be tolerated. For example, these substitutions may be in regions of the Proteins that are not critical for activity. The analysis of the crystal structure of a protein, and structure modeling of protein based software, can be used to identify regions of a protein that can be modified (using the site-directed mutagenesis, recomposition, etc.) to change real way the properties and / or increase the functionality of the protein.

You can also change various properties and 3 D protein target characteristics without affecting Adverse protein activity / functionality. The conserved amino acid substitutions can be expected to be tolerated / do not adversely affect the configuration of three molecule dimensions. The amino acids can be located in the following classes: non-polar, polar no load, Basic, and acid. Conservative substitutions for which a amino acid of one class is replaced with another amino acid of the same type falls within the scope of the present invention while the Substitution is not adverse to the biological activity of the compound. Table 1 provides a list of examples of amino acids that It belongs to each class.

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TABLE 1

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4

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In some cases, they can also be done non-conservative substitutions The critical factor is that you are substitutions should not decrease significantly from the Functional activity / biological activity of the toxin.

As used herein, reference to "isolated" polynucleotides and / or "purified" toxins refers to these molecules when they are not associated with the other molecules with which they would bind in nature. Thus, the reference to "isolated" and / or "purified" means the implication of the "hand of man" as described herein. For example, a bacterial toxin "gene" of the present invention placed in a plant for expression is an "isolated polynucleotide." Similarly, a Paenibacillus protein, exemplified herein, produced by a plant is an "isolated protein."

Due to code degeneracy / redundancy genetic, a variety of different DNA sequences can be encode the amino acid sequences described herein document. Those skilled in the art know well how to create sequences of Alternative DNAs encoding the same or essentially the same toxins These variant DNA sequences are described.

Sequence optimization for expression in plants . In order to obtain a high expression of heterologous genes in plants, it may be preferred to genetically modify said genes so that they are more effectively expressed in (the cytoplasm of) plant cells. Corn is one such plant where it may be preferable to redesign the heterologous gene (s) before transformation to increase its level of expression in said plant. Therefore, an additional stage in the design of genes encoding a bacterial toxin is modified again by genetic engineering of a heterologous gene for optimal expression.

One reason to genetically modify a bacterial toxin for expression in corn is due to the non-optimal G + C content of the native gene. For example, the very low G + C content of many native bacterial genes (and later biased towards high A + T content) results in the generation of sequences that mimic or duplicate the gene control sequences of known plants. which are highly rich in A + T. The presence of some sequences rich in A + T with the DNA gene (s) introduced into plants ( for example, regions of TATA cells normally found in gene promoters) may result in aberrant transcription of the (of the) gene (s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA ( for example, depololdenylation signal sequences (AAUAAA), or complementary sequences with the small nuclear RNAs involved in the aid of preRNA) can lead to RNA instability. . Therefore, an objective in the design of genes encoding a bacterial toxin for the expression of corn, more preferably referred to as a plant optimized gene (s), is to generate a DNA sequence that has a higher G + C content, and preferably one close to the corn genes encoding metabolic enzymes. Another objective in the design of the optimized gene (s) of plants encoding a bacterial toxin is to generate a DNA sequence in which sequence modifications do not prevent
translation.

The Table below (Table 2) how tall is the content of G + C in corn. For the data in Table 2, which encode the regions of the genes were extracted from the entries of GenBank (Release 71), and base compositions were calculated using the MacVector.TM program. (IBI, New Haven, Conn.). The intron sequences were ignored in the calculations.

Due to the plasticity produced by the redundancy / degeneracy of the genetic code (i.e. some amino acids are specified by more than one codon), evolution of genomes in organisms or different kinds of organisms has given as a result a differential use of redundant codons. This "codon deviation" is reflected in the base composition average of regions that encode proteins. For example, organisms with relatively low G + C contents use codons that they have A or T in the third position of redundant codons, while those with older G + C content use codons that have G or C in the third position. It is believed that the presence of "minor" codons within an mRNA can reduce the absolute translation speed of that mRNA, especially when the relative abundance of the loaded tRNA corresponding to the Lower codon is low. An extension of this is that the decrease in the translation speed for individual minor codons would be at least additive for multiple codons. Therefore, mRNAs that they have high relative contents of lower codons would have relatively low translation speeds. This speed is would reflect by lower subsequent levels of the protein coded

In genetic engineering remodification of the genes that encode a bacterial toxin for expression in corn (or another plant, such as cotton or soybeans), the deviation of the codon of the plant. The codon deviation for corn is the distribution of the statistical codon that the plant uses to encode your proteins and the use of preferred codon is shown in Table 3. After determining the deviation, the percentage of codon frequency in the gene (s) of interest. Primary codons preferred by the plant should be determine as well as the second and third choice of codons preferred. After this, the amino acid sequence of the Bacterial toxin of interest is reversed in a reverse manner  that the resulting nucleic acid sequence encodes exactly the same protein as the native gene that is desired to be expressed from heterologous way. The new DNA sequence is designed using the codon deviation information so that it corresponds to the Most preferred codons of the desired plant. The new one sequence is then analyzed to determine enzyme sites of restriction that could have been created by the modification. The identified sites are also modified by replacing the codons with the second or third preferred choice of codons. Other sites in the sequence that could affect transcription or translation of the gene of interest are exon junctions: intron (5 'or 3 '), poly A addition signals, or termination signals of the RNA polymerase. In addition, the sequence is analyzed and modified to reduce the frequency of TA or GC doublets. In addition to the doublets, the blocks of sequence G or C that have more than about four residues that are the same can affect the transcription of the sequences. Therefore, these blocks are also modified replacing the codons of the first or second choice, etc., with the following preferred codon of choice.

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TABLE 2

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5

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It is preferred that the gene (s) encoding (n) a bacterial toxin contains (n) approximately 63% of the first codon choice, between approximately 22% and approximately 37% of the second election of codons, and between about 15% and about 0% of the third choice of codons, where the total percentage is 100%. The more preferred is that the gene (s) contain approximately 63% of the first codon choice, at least approximately 22% of the second codon choice, approximately 7.5% of the third choice of codons, and approximately 7.5% of the fourth choice of codons, where the Total percentage is 100%. The use of the preferred codon for Genetic engineering modification of genes for expression in corn is shown in Table 3. The procedure described previously allows those skilled in the art to modify the gene (s) that are strangers for a plant particularly so that genes are optimally expressed in plants. The procedure is further illustrated in the PCT WO application 97/13402.

In order to design the optimized genes of plants encoding a bacterial toxin, the amino acid sequence of said protein is inversely translated into a DNA sequence that uses a non-reduced genetic code from a codon deviation table for the sequences. of genes for the particular plant, as shown in Table 2. The resulting DNA sequence, which is completely homogeneous in codon usage, is further modified to establish a DNA sequence that, in addition to having a greater degree of diversity codon, it also contains strategically placed enzyme recognition sites, desirable base composition, and a lack of sequences that could interfere with gene transcription, or mRNA translation
product.

TABLE 3

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6

Thus, the synthetic genes that are functionally equivalent to the toxins of the present invention They can be used to transform guests, including plants. The additional guidance regarding the production of synthetic genes you can find, for example, U.S. Patent No. 5,380,831.

In some cases, especially during expression in plants, it may be advantageous to use truncated genes that express truncated proteins. Höfte et al . 1989, for example, described in the background section above, described protoxin and core toxin segments of B toxins. t . Preferred truncated genes will typically encode 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, S0, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of the full length toxin.

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GM guests . The genes encoding toxins as described can be introduced into a wide variety of microbial or plant hosts. In preferred embodiments, the cells of transgenic plants and plants are used. Preferred plants (and plant cells) are oats, corn and cotton.

In preferred embodiments, the expression of a toxin gene results, directly or indirectly, in the intracellular production (and maintenance) of pesticidal proteins. Plants can become resistant to insects in this way. When, the transgenic / recombinant / transformed / transfected host cells (or their contents) are ingested by the pests, the pests will ingest the toxins. This is the preferred way that causes pest contact with the toxin. The result is the control (death or sickness) of the plague. Sucking pests can also be controlled in a similar way. Alternatively, suitable microbial hosts can be applied, for example Pseudomonas such as P. fluorescens , they can be applied when the target pests are present, the microbes can proliferate there, and are ingested by the target pests. The microbe that harbors the toxin gene can be treated under conditions that prolong the activity of the toxin and stabilize the cell. The treated cell, which maintains toxic activity, can then be applied to the target pest environment.

When the toxin gene is introduced by a suitable vector in a microbial host, and said host is applied to the environment in a living state, certain microbes should be used Guest. Microorganism hosts are selected so that occupy the "phytosphere" (phylloplane, philosopher, rhizosphere, and / or rizoplano) of one or more crops of interest. These microorganisms are selected so that they are able to compete in a way successful in the particular environment (crop habitat and others insects) with wild-type microorganisms, provide the maintenance and stable expression of the gene that expresses the polypeptide pesticide, and, desirably, provide an improvement in pesticide protection from degradation and inactivation environmental.

A large number of microorganisms that disable phylloplane (the surface of the leaves of plants) and / or the rhizosphere (the soil surrounding the roots of the plant) of a wide variety of important crops are known. These microorganisms include bacteria, algae, and fungi. Of particular interest are microorganisms, such as bacteria, for example, the genera Pseudomonas, Erwinia, Serratia, Klebsiella , Xanthomonas, Streptomyces, Rhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, Azotobacter, fungi, particularly yeasts, for example, the genera Saccharomyces, Cryptococcus , Kluyveromyces , Sporobolomyces, Rhodotorula , and Aureobasidium . Of particular interest are species of phytosphere bacteria such as Pseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens, Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonas spheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus, and Azotoiiii ; and photosphere yeast species such as Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans . Also of interest are pigmented microorganisms.

Insertion of genes to form transgenic hosts . The transformation / infection of plants, plant cells and other host cells with polynucleotides is described as described expressing proteins of the present invention. Plants transformed in this way can become resistant to attack by the target pest (s).

A variety of procedures to introduce a gene that encodes a protein pesticide in the target host under conditions that allow stable maintenance and gene expression. These procedures they are well known to those skilled in the art and are described, for example, in U.S. Patent No. 5,135,867.

For example, a large number of cloning vectors comprising a replication system in E. coli and a marker that allows the selection of transformed cells are available for the preparation of foreign gene insertion in higher plants. The vectors comprise, for example, the pBR322 series, pUC, M13mp series, pACYC184, etc. In accordance with the above, the sequence encoding the toxin can be inserted into the vector at a suitable restriction site. The resulting plasmid is used for transformation into E. coli . E. coli cells are grown in a suitable nutrient medium, then harvested and lysed. The plasmid is recovered. Sequence analysis, restriction analysis, electrophoresis, and other biochemical-molecular-biological procedures are generally carried out as analysis procedures. After each manipulation, the DNA sequence used can be cleaved and attached to the following AND sequence. Each plasmid sequence can be cloned into the same or another plasmid. Depending on the insertion procedure of the desired genes in the plant, other DNA sequences may be necessary. If, for example, the Ti or Ri plasmid is used for the transformation of the plant cell, then at least the right edge, but often the right and left edge of the T-DNA of the Ti or Ri plasmid, has to be attached to the flanking region of the genes to be inserted. The use of T-DNA for the transformation of plant cells has been re-investigated and intensively described in EP 120 516; Hoekema (1985) in: The Binary Plant Vector System, Offset-durkkerij Kanters BV, Alblasserdam, chapter 5; Fraley et al ., Crit. Rev. Plant Sci. 4: 1-46; and An et al . (1985) EMBO J. 4: 277-287.

A large number of techniques are available to insert DNA into a plant host cell. Those techniques include transformation with T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transformation, fusion, injection, biolistic agent (microparticle bombardment), or electroporation as well as other possible procedures. If Agrobacteria are used for transformation, the DNA to be inserted must be cloned into special plasmids, namely in an intermediate vector or in a binary vector. Intermediate vectors can be integrated into the Ti or Ri plasmid by homologous recombination due to the sequences that are homologous to the sequences in the T-DNA. The Ti or Ri plasmid also comprises the vir region necessary for T-DNA transfer. Intermediate vectors cannot replicate themselves in Agrobacteria. The intermediate vector can be transferred into Agrobacterium tumefaciens by an auxiliary plasmid (conjugation). Binary vectors can replicate themselves in both E. coli and Agrobacteria . They comprise a selection marker gene and a linker or polylinker that are framed by the right and left T-DNA border regions. They can be transformed directly into Agrobacteria (Holsters et al . [1978] Mol. Gen. Genet. 163: 181-187). The Agrobacterium used as a host cell is to comprise a plasmid that carries a vir region. The vir region is necessary for the transfer of T-DNA in the plant cell. Additional T-DNA may be contained. The bacterium thus transformed is used for the transformation of plant cells. Plant explants can be grown advantageously with Agrobacterium tumefaciens or Agrobacterium rhizogenes for DNA transfer in the plant cell. The whole plants can then be regenerated from infected plant material (for example, leaf parts, stem segments, roots, but also protoplasts or cells grown in suspension) in a suitable medium, which may contain antibiotics or biocides for selection. The plants thus obtained can then be tested to determine the presence of the inserted DNA. No special demand for plasmids is made in the case of injection and electroporation. It is possible to use ordinary plasmids, such as, for example, derivatives of pUC.

Transformed cells grow inside of plants in the usual way. They can form germ cells and transmit the character (s) to the plants of progeny. Such plants can be developed in the normal way and cross with plants that have the same hereditary factors transformed or other hereditary factors. Individuals resulting hybrids have phenotypic properties corresponding.

The genes that encode the bacterial toxin are can express from transcription units inserted in The genome of plants. Preferably, said units of transcription are recombinant vectors capable of integration stable in the genome of plants and allow the selection of lines transformed vegetables that express mRNA encoding the proteins

Once the DNA inserted into the genome has been integrated, it is relatively stable there (and does not come out again). It usually contains a selection marker that gives transformed plant cells resistance to a biocide or an antibiotic, such as kanamycin, G418, bleomycin, hygromycin, or chloramphenicol, inter alia . The marker used individually should in accordance with the foregoing allow the selection of transformed cells instead of cells that do not contain the inserted DNA. The gene (s) of interest are preferably expressed either by constitutive or inducible promoters in the plant cell. Once it has been expressed, the mRNA is translated into proteins, thereby incorporating the amino acids of interest in the protein. The genes encoding a toxin expressed in plant cells may be under the control of a constitutive promoter, a tissue specific promoter, or an inducible promoter.

There are several techniques for introducing foreign recombinant vectors into plant cells, and for obtaining plants that maintain and stably express the introduced gene (US Pat. Nos. 4,945,050 to Cornell and 5,141,131 to DowElanco, now Dow AgroSciences, LLC). In addition, plants can be transformed using Agrobacterium technology, see US Patent No. 5,177,010 to the University of Toledo; 5,104,310 of Texas A&M; European Patent Application 0131624B1; European Patent Application 120516, 159418B1 and 176,112 of Schilperoot; U.S. Patents Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot; European Patent Applications 116718, 290799, 320500 all of Max Planck; European Patent Applications 604662 and 627752, and US Patent No. 5,591,616, of Japan Tobacco; European Patent Applications 0267159 and 0292435, and United States Patent No. 5,231,019, all of Ciba Geigy, now Novartis; United States Patents Nos. 5,463,174 and 4,762,785, both of Calgene; and United States Patents Nos. 5,004,863 and 5,159,135, both of Agracetus. Another transformation technology includes pin technology. See US Patents Nos. 5,302,523 and 5,464,765, both of Zeneca. Electroporation technology has also been used to transform plants. See WO 87/06614 of the Boyce Thompson Institute; United States Patents Nos. 5,472,869 and 5,384,253, both of Dekalb; and WO 92/09696 and WO 93/21335, both of Plant Genetic Systems. In addition, viral vectors can also be used to produce transgenic plants that express the protein of interest. For example, monocotyledonous plants can be transformed with a viral vector using the procedures described in US Pat. Nos. 5,569,597 to Mycogen Plant Science and Ciba-Giegy, now Novartis, as well as US Pat. Nos. 5,589,367 and 5,316,931, both from Biosource.

As mentioned above, the way in which the DNA construct is introduced into the host plant is not critical. Any procedure that provides effective transformation can be used. For example, various methods for transforming plant cells are described herein and include the use of Ti or Ri plasmids and the like to perform Agrobacterium- mediated transformation. In many cases, it will be desirable for them to have the construction used for the transformation that borders one or both sides by T-DNA edges, more specifically the right edge. This is particularly useful when the construction uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode of transformation, although T-DNA edges may find use with other modes of transformation. When Agrobacterium is used for the transformation of plant cells, a vector that can be introduced into the host can be used for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. The introduction of the vector can be performed by electroporation, tri-parental pairing and other techniques to transform gram-negative bacteria than those known to those skilled in the art. The medium of the transformation vector in the Agrobacterium host is not critical. The Ti or Ri plasmid containing the T-DNA for recombination may be capable of or unable to cause galling, and is not critical as long as the vir genes are present in said host.

In some cases when Agrobacterium is used for transformation, the construction of the expression that is within the borders of T-DNA will be inserted into a high-spectrum vector such as pRK2 or its derivatives as described in Ditta et al ., ( PNAS USA (1980) 77: 7347-7351 and EPO 0 120 515, which are incorporated herein by reference, included within the expression construct and the T-DNA will be one or more markers as described herein. which allows the selection of transformed Agrobacterium and transformed plant cells.The particular marker employed is not essential, depending on the preferred marker of the host and construction used.

For the transformation of plant cells using Agrobacterium , explants can be combined and incubated with the transformed Agrobacterium for sufficient time to allow their transformation. After transformation, the Agrobacteria are killed by selection with the appropriate antibiotic and the plant cells are grown with the appropriate selective medium. Once calluses are formed, bud formation can be stimulated using appropriate plant hormones according to the procedures well known in the art of plant tissue culture and plant regeneration. However, an intermediate phase of callus is not always necessary. After sprout formation, said plant plants can be transferred to the medium that stimulates root formation thereby completing plant regeneration. The plants can then be grown to seed and such seed can be used in the establishment of future generations. Regardless of the transformation technique, the gene encoding a bacterial toxin is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell including in the vector a plant promoter regulatory element, as well as transcription termination regions not translated into 3 'such as Nos and
Similar.

In addition to the numerous technologies for plant transformation, the type of tissue that is put into Contact with foreign genes may vary as well. Such fabric would include but not be limited to embryogenic tissue, tissue of callus types I, II, and III, hypocotyl, meristem, root tissue, tissues for expression in the phloem, and the like. Almost all the plant tissues can be transformed by dedifferentiation using appropriate techniques described in the present document

As mentioned above, you can use a variety of markers that can be selected, if want. The preference of a marker is at the discretion of the skilled in the art, but any of the markers that you can select can be used together with any other gene not listed in this document that could work as a bookmark that can be selected. Such markers that can be select include but are not limited to aminoglycoside gene Tn5 transposon phosphotransferase (Aph II) encoding the antibiotic resistance kanamycin, neomycin and G418 as well such as those genes that encode resistance or tolerance to glyphosate herbicides; hygromycin; methotrexate; phosphinothricin (bialafos); imidazolinones, sulfonylureas and triazolopyrimidine, such as clorsulfuron; Bromoxynil, dalapon and the like.

In addition to a selectable marker, it may be desirable to use an indicator gene. In some cases an indicator gene can be used with or without a marker that can be selected. Indicator genes are genes that are typically not present in the body or recipient tissue and typically encode proteins that result in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Wising et al . Ann. Rev. Genetics, 22, 421 (1988). Preferred reporter genes include the beta-glucuronidase (GUS) of the E. coli uidA locus, the E. coli Tn9 chloramfenicol acetyl transferase gene , the fluorescent green protein of the bioluminescent jellyfish Aequorea victoria, and the luciferase genes of the firefly Photinus pyralis . An assay to detect expression of the reporter gene can then be carried out in a suitable time after said introduction has been introduced into the recipient cells. One such preferred assay involves the use of the gene encoding beta-glucuronidase (GUS) of the E. coli uidA locus as described by Jefferson et al ., (1987 Biochem. Soc. Trans. 15, 17-19) to identify cells transformed.

In addition to the regulatory elements of plant promoters, regulatory elements of promoters from a variety of sources can be used effectively in plant cells to express foreign genes. For example, the regulatory elements of promoters of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as cauliflower mosaic virus (35S and 19S), 35T (which is a promoter remodified by 35S genetic engineering, see US Patent No. 6,166,302, especially example 7E) and the like can be used Regulatory elements of the plant promoter include but are not limited to the small subunit of ribulose-1,6-bisphosphate (RUBP) carboxylase (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, promoters thermal shock, and tissue specific promoters. Other elements such as matrix binding regions, scaffold binding regions, introns, enhancers, polyadenylation sequences and the like may be present and thus, can improve the efficiency of DNA transcription or integration. Such elements may or may not be necessary for DNA function, although they may provide better expression or functioning of the DNA affecting transcription, mRNA stability, and the like. Such elements may be included in the DNA as desired to obtain the optimal embodiment of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, Adhintron 6, the guiding sequence of the alfalfa mosaic virus coating protein, the guiding sequence of the corn scratch virus coating protein, as well as others available by those skilled in the art. The regulatory elements of the constitutive promoters can therefore also be used by directing the expression of the continuous gene in all cell types and at all times (for example, actin, ubiquitin, CaMV 35S, and the like). The regulatory elements of tissue-specific promoters are responsible for gene expression in specific cell types or tissues, such as leaves or seeds (e.g., zein, oleosin, napine, ACP, globulin and the like) and these can also be used. .

The regulatory elements of the promoters they can also be active during a certain stage of development of plants as well as active in plant tissues and organs. The Examples of such include but are not limited to items. regulators of pollen-specific promoters, specific to embryo, corn silk specific, fiber specific cotton, root specific, seed endosperm specific  and the like In certain circumstances it may be desirable to use a regulatory element of the promoters, which is responsible for the gene expression in response to a specific signal, such as a physical stimulus (heat shock genes), light (RUBP carboxylase), hormone (Em), metabolites, chemical compound, and stress. Can be use other desirable transcription and translation elements that They work in plants. Numerous vectors are known in the art. of transfer of plant-specific genes.

Molecular biology techniques can be used to clone and sequence the toxins described herein. Additional information can be found in Sambrook, J., Fritsch, EF, and Maniatis, T. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, which is incorporated herein by
reference.

Resistance Management With the increased commercial use of insecticidal proteins in transgenic plants, a consideration is resistance management. That is, there are numerous companies that use Bacillus thuringiensis toxins in their products, and there is a matter about insects that develop resistance to B toxins. t .. A strategy for resistance management would be to combine the toxins from TC toxins produced by Xenorhabdus, Photorhabdus , and the like with toxins such as B. t ., crystalline toxins, soluble insecticidal proteins from Bacillus strains ( see, for example, documents WO98 / 18932 and WO99 / 57282), or other insect toxins. The combinations can be formulated for a sprayable application or they can be molecular combinations. Plants can be transformed with bacterial genes that produce two or more different insect toxins (see, for example, Gould, 38 Bioscience 26-33 (1988) and U.S. Patent No. 5,500,365; similarly, the application European Patent 0 400 246 A1 and United States Patents Nos. 5,866,784; 5,908,970; and 6,172,281 also describe the transformation of a plant with two crystalline toxins of B. t .). Another method of producing a transgenic plant that contains more than one insect resistance gene would be to first produce two plants, each plant containing a gene insect resistance gene. These plants can then be crossed using traditional plant improvement techniques to produce a plant that contains more than one insect resistance gene. Thus, it should be clear that the phrase "comprising a polynucleotide" as used herein means at least one polynucleotide (and possibly more, contiguous or not) unless specifically indicated otherwise.
way.

Formulations and other distribution systems . The formulated bait granules containing spores and / or crystals of the Paenibacillus subject isolation, or recombinant microbes comprising the genes that can be obtained from the isolation described herein, can be applied to the soil. The formulated product can also be applied in the form of a seed coating or root treatment or total plant treatment in the late stages of the crop cycle. Plant and soil cell treatments can be used in the form of powders or granules or wettable powders, mixing with various inert materials, such as organic minerals (phyllosilicates, carbonates, sulfates, phosphates, and the like) or botanical materials (corn cobs powder, rice husks, nutshells, and the like). The formulations may include dispersion-adhesion adjuvants, stabilizing agents, other pesticidal additives, or surfactants. Liquid formulations can be in aqueous or non-aqueous base and used as foams, gels, suspensions, emulsifiable concentrates, or the like. The ingredients may include rheological agents, surfactants, emulsifiers, dispersants, or polymers.

As those skilled in the art will appreciate, the Pesticide concentration will vary widely depending on the nature of the particular formulation, particularly if it is a concentrated or will be used directly. The pesticide will be present in at least 1% by weight and can be 100% by weight. The Dry formulations will have between about 1-95% by weight of pesticide while liquid formulations in general will be between about 1-60% by weight of the solids in the liquid phase. Formulations in general will have between about 10 2 and about 10 4 cells / mg. These formulations will be administered at approximately 50 mg. (liquid or dry) up to 1 kg or more per hectare.

The formulations can be applied to the pest environment, for example, soil and foliage, by sprinkling, sprinkling, or the like.

Another distribution scheme is the incorporation of the genetic material of the toxins in a baculovirus vector. Baculoviruses infect hosts of particular insects, including those directed desirably with the toxins. The infectious baculovirus that harbor an expression construct for toxins can be introduced into areas of infestation of insects to poison therefore or poison insects infected.

Insect viruses, or baculovirus, is known that infect and reverse certain insects. The Virus or insect involvement is slow, and viruses do not stop Insect feeding immediately. In this way, viruses they are not contemplated to be optimal as pest control agents of insects However, the combination of toxin genes in a baculovirus vector can provide an effective way of transmit toxins In addition, since different baculovirus are specific for different insects, it may be possible to use a particular toxin to selectively direct particularly damaging insect pests. A particularly useful vector To toxin genes is the nuclear polyhedrosis virus. The transfer vectors using this virus have been described and are now the vectors of choice to transfer foreign genes to insects The recombinant of the virus toxin gene can be build in a way that can be transmitted orally. The baculovirus normally infect insect victims through of the intestinal mucosa of the middle intestine. The toxin gene inserted behind a coating protein promoter Strong viral would be expressed and should quickly kill the insect infected.

In addition to an insect virus or baculovirus or plant distribution system for toxins of the proteins of the present invention, proteins can be encapsulated using Bacillus thuringiensis encapsulation technology such as but not limited to US Pat. 4,695,455; 4,695,462; 4,861,595 which are all incorporated herein by reference. Another distribution system for the protein toxins of the present invention is the formulation of the protein in a bait matrix, which can then be used above and below ground insect bait stations. Examples of such technology include but are not limited to PCT patent application WO 93/23998, which is incorporated herein by
reference.

Systems based on plant RNA viruses They can also be used to express bacterial toxin. Doing this, the gene that encodes a toxin can be inserted in the region of the coating promoter that will infect the host plant of interest. The toxin can then be expressed by providing this way the protection of the plant from insect damage. The systems based on plant RNA viruses are described in the Patents of United States numbers 5,500,360 to Mycogen Plant Sciences, Inc. and U.S. Patents Nos. 5,316,931 and 5,589,367 of Biosource Genetics Corp.

In addition to producing a transforming plant, there are other distribution systems where it may be desirable Genetically re-modify the bacterial gene (s). For example, a protein toxin is can build by fusion together with a molecule attractive to insects as a food source with a toxin. After purification in the laboratory such agent Toxic with bait "built inside" can be packaged inside of conventional insect trap housings.

Mutants DAS1529 isolation mutants of the invention can be prepared by methods that are well known to those skilled in the art. For example, an asporogenic mutant can be obtained by ethyl methane sulphonate (EMS) mutagenesis of an isolation. The mutants can be made using ultraviolet light and nitrosoguanidine by methods well known in the art.

Below are the examples that illustrate Procedures for practicing the invention. These examples are not They should consider as limiting. All percentages are in weight and all solvent mixing ratios are in volume unless otherwise indicated.

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Example one

Isolation and discovery of the insecticidal activity of DAS1529 as a Paenibacillus sp

A bacterial strain, named DAS1529, was found to produce factors that were growth inhibitors of lepidopteran insect neonates, corn cob worm ( Heliothis zea; CEW), tobacco yolk worm ( Heliothis virescens; TBW), and Horned tobacco worm ( Manduca sixth; THW).

DAS 1529 was grown in 2% Protease medium Peptone No. 3 (PP3) (Difco Laboratories, Detroit, MI) supplemented with 1.25% NaCl or JB medium supplemented with 0.2% glucose. He Bacterial culture was grown at 25 ° C for ~ 40 hours at 150 rpm

Insecticide-active factors were initially found in the fermentation broth that was concentrated on 5 kDa molecular weight shear filters. Factors that were heat labile (inactivated by heating at 85 ° C for 20 minutes). These data indicated that the factors were proteinaceous. See also the end of Example 4.

To identify the active factors in the cell sediments, the bacterial culture was centrifuged at 8000 rpm at 4 ° C for 15 minutes, washed once with distilled water sterile, and resuspended at 33X of the culture volume original in sterile distilled water, and subsequently to bioassay of insects as described later in Example 3. The data of bioassay for strain DAS 1529 is summarized in Table 4. The data showed that the culture broth and bacterial cells DAS 1529 concentrates conferred good activity against CEW (30 a 50% mortality at 33X) and TBW (100% mortality at 33X). Those toxin factors in DAS 1529 had a relative significance for the development of commercial transgenic products that they direct  Lepidoptera insects (for example, CEW and TBW) in corn and cotton.

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8

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Example 2

Classification of DAS 1529

Molecular phylogeny was performed to determine the taxonomic affiliation of strain DAS 1529. The 16S rDNA nucleotide sequence of DAS 1529 is determined and used for similarity and phylogenetic analysis (using kit ABI MicroSeq Kit). The sequence is provided as SEQ ID NO: 16. The BLAST research results are as follows:

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10

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These same top scoring sequences of BLAST research were also compared using the routine Gap (Needleman and Wunsch, J. Mol. Biol. 48; 443-453 (1970)) of GCG version 10.2, with the following results:

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A number of related sequences, which include the upper scoring sequences indicated above, were also trimmed and aligned as described by Shida et al . (Int. J. Syst. Bacteriol. 47: 289-298, 1997), using the CLUSTAL W sequence alignment program (Thompson, JD, DG Higgins, and TJ Gibson, Nucleic Acids Res. 22: 4673-4680, 1994 ). The results clearly place DAS1529 in the Paenibacillus popilliae / Paenibacillus lentimorbus subgroup of the Paenibacillus genus identified by Pettersson et al . ( Int . J. Syst . Bacteriol . 49: 531-540, 1999), and are consistent with the analyzes outlined above. This subgroup includes the species associated with insects P. popilliae and P. lentimorbus , as well as P. thiaminolyticus, Paenibacillus sp. T-168 and C-168, and " Bacillus tipchiralis ", which is not known to have an association with insects (Pettersson et al ., 1999). As indicated by Wayne et al . (Int. J. Syst. Bacteriol. 37: 463-464, 1987) and Vandamme et al . (Microbiol. Rev. 60: 407-438), rDNA sequences that are greater than 97% identity cannot generally be used to assign a bacterial strain to a particular species in the absence of additional information. In the case of DAS1529, the insecticidal activity on lepidoptera and evidence of a thiaminase are not consistent with P. popilliae and P. known lentimorbus , and the association of insects is not consistent with P. known thiaminolyticus (as well as the other species of the subgroup).

As other strains of Paenibacillus are known as causative agents of milky bacteriosis in Japanese beetle larvae ( Popillia jalonica ; Harrison et al., 2000), DAS 1529 was tested for activity on June beetles, a relative of Japanese beetles . No activity was found for crops grown in JB and PP3 medium. Microscopic examination of the cultures revealed similarly colored canes without visible sporulation or parasporal crystals present. The inventors are able to show that DAS 1529 can sporulate in defined medium and culture conditions and within the sixth Manducca hemolymph. Paenibacillus strains active against the Japanese beetle are known to be typically associated with paraspora and parasporal bodies (Harrison et al., 2000).

Additional work will be needed to determine if DAS 1529 belongs or should be assigned to a new species denomination

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Example 3

Insect bioassay methodology

Two bioassay procedures of insects to get the results presented later. A 96-well format and yb 128-well format were used to the primary selection to determine the activity against insects of insect lepidoptera. A diet incorporation format of 24 wells were used to determine the specific activity 24- (CL 50) of the toxin.

For the 96-well format, artificial diet was dispensed in 96-well microtiter plates. Each well averaged approximately 0.32 cm and contained 150 µl of artificial diet. Samples / toxins were applied at a rate of 50 µl / well for fermentation broth, cell pellets, and purified toxins. Positive control ( Cry 1Ac) at appropriate doses and negative controls (water, medium blank, bacterial host strains that do not express the target toxin) at a higher dose were included. The samples were allowed to dry for approximately 1-3 hours so that the samples lost their moisture but the diet maintained its humidity. Some insect eggs were dispersed on the surface of the sample-treated diet, or an individual insect larva was seeded per well. The infested plate was sealed either with mylar coating on iron or covered with adherent caps with perforations. Tiny air holes were made in the mylar coating to ensure the air supply to the insects. Plates were incubated at 28 ° C for 5 days and scored to determine mortality and atrophy. This was done on a per-well basis, ignoring the number of larvae per well, as multiple eggs are often laid per well. Activity scores were then assigned to each treatment: 0 = no activity, healthy larvae such as water control wells, 1 = larvae were atrophied, or atrophied with some mortality, 2 = all larvae were dead.

The specific activities (CL_ {50}) of samples / toxins were determined by bioassay of incorporation of diet in Nutrend trays of (Nu-Trend? Container Corp., Jacksonville, FL). The artificial insect diet was performed just before using and keep in a liquid state at 55ºC in a water bath. They were made serial dilutions (≥ 5) mixing 27 ml of artificial diet with no more than 3 ml of samples / toxins. A total of 30 ml of sample and diet mixture was stirred in a vortex apparatus for 30 seconds and then distributed equally in each tray, filling sim 50% of the volume of the well. The trays were allowed to chill for at least 30 minutes before infestation. Be infected an insect test in each well, and mylar was sealed transparent over the top of each tray so that contain insects Small holes were drilled with a insect pin in the mylar on each well for air circulation. In general the tests were kept at 25 ° C for 6 days but some may have been kept at 30 ° C for 4 days if faster results are needed. A set of positive and negative controls were developed for each activity. The trials were classified based on mortality but Atrophy data was not recorded. The procedures statistics were used to estimate the values of CL 50 for the samples tested and expressed as ng or \ mug / ml of diet.

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Example 4

Purification and biochemical characterization of toxins fermentation broth insecticides - thiaminase from DAS1529

The fermentation broths of DAS 1529 contained insecticidal activity against lepidoptera species, such as tobacco worm worm, corn cob worm, and fleshy tobacco worm. The nature of the activity insecticide was investigated by biochemical purification and characterization. The corn cob worm bioassay, as described in Example 3, it was used during the procedure of purification to follow insecticidal activities.

The fermentation broths of DAS 1529 are produced using 2% PP3 supplemented with 1.25% NaCl and processed as described in Example 1. Four were concentrated liters of broth using a spiral ultrafiltration cartridge Amicon (Beverly, MA) Type S 1 and 10 (molecular weight cut of 10 kDa) attached to an Amicon filtration device M-12 according to the recommendations of the maker. The retentate was diafiltered with 20 mM sodium phosphate, pH 7.0 (Buffer A) and applied at 5 ml / min to an exchange column anionic Q cepharose XL (1.6 x 10 cm). The column was washed with 5 Buffer bed volumes A to remove unbound proteins. The toxin activity was eluted by 1.0 M NaCl in buffer TO.

The fraction that contained the activity insecticide was loaded in 20 ml aliquots on a column of Macro-Prep SE1000 / 40 gel filtration (2.6 x 100 cm) which was equilibrated with buffer A. The protein was eluted in buffer A at a flow rate of 3 ml / min. Fractions with activity against Corncob worm were applied to a MonoQ column (1.0 x 10 cm) balanced with 20 mM Tris-HCl, pH 7.0 (Buffer B) at a flow rate of 1 ml / min. The proteins bound to the column were flowed with a linear gradient of 0 to 1 M NaCl in Buffer B at 2 ml / min for 60 min. Fractions of two were collected milliliters and the activity was determined as described in the Example 1.

Solid (NH 4) 2 SO 4 was added to the previous active protein fractions up to a final concentration of 1.7 M. The proteins were then applied to a column of phenyl-Superose (1.0 x 10 cm) balanced with 1.7 M (NH 4) 2 SO 4 in 50 mM of potassium phosphate buffer, pH 7 (Buffer C) at 1 ml / min. After wash the column with 10 milliliters of Buffer C, bound proteins to the column were flowed with a linear gradient of Buffer C to 5 mM potassium phosphate, pH 7.0 at 1 ml / min for 120 min. The majority of the active fractions determined by bioassay gathered and dialyzed all night against the Tampon TO.

The dialyzed sample was applied to a column of Mono Q (0.5 x 5 cm) that was equilibrated with Buffer B at 1 ml / min. The column bound proteins were eluted at 1 ml / min by a Linear gradient from 0 to 1 M NaCl in Buffer B. Fractions active met and adjusted to a final concentration of (NH 4) 2 SO 4 of 1.7 M. Proteins are then applied to a phenyl-Superose column (0.5.0 x 5 cm) equilibrated with Buffer C at 1 ml / min. The proteins bound to the column were flowed with a linear gradient of Buffer C at 5 mM of potassium phosphate, pH 7.0 at 0.5 ml / min for 40 min. The purified fractions were collected and dialyzed throughout one night against Tampon A.

The molecular weight of the final purified toxin was examined by a Superdex gel filtration column S-200 The toxin showed a native molecular weight of approximately 40 kDa. SDS-PAGE of toxins purified showed a predominant band of approximately 40 kDa. This suggested that the native DAS 1529 toxin (in this fraction) was a monomer of approximately 40 kDa.

The purified toxin was electrophoresed in 4-20% SDS-PAGE and immunoblotted to a PVDF membrane. The transfer was subjected to an amino acid analysis and N-terminal amino acid sequencing (SEQ ID NO. 17). Investigation of the protein database (NCBI-NR) using the sequence as a doubt revealed that it is 95% identical to the approximately 42 kDa thiaminase I of Bacillus thiaminolyticus (Campobasso et al ., 1998; GENBANK No. 2THIA access; SEQ ID NO: 18). Partial sequence alignments are illustrated in Figure 3, which would have the same alignment with GENBANK Accession No. AAC44156 (precursor to thiaminase I; U17168 is the corresponding entry in GENBANK for DNA, which could be expressed to achieve a thiaminase produced and secreted from the bacterial cell). The purified thiaminase from DAS 1529 was tested on corn cob worm (CEW), the results are shown in Figure 4. This toxin did not kill the corn cob worm (up to a concentration of 8 mg / cm2). ) but showed a 95% growth inhibition at a concentration as low as 5 ng / cm2. It was also found that purified thiaminase was not deactivated by proteinase K.

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Example 5

Cloning of genes that encode insecticidal factors produced by DAS 1529

In an attempt to clone the (s) nucleotide sequence (s) encoding the produced factor (s) DAS 1529, a cosmid library using DNA isolated from DAS 1529 and it selected for insecticidal activity. Six were identified recombinant cosmid clones that produced insecticidal activity  against the neonates of the corn cob worm and the Tobacco bud Three of the cosmid clones produced factors heat labile (when heated at 85 ° C for 20 minutes) which They resulted in insect mortality. The other three clones of cosmids produced heat labile factors that were inhibitors  of growth for insects. One of the cosmids that produced insect mortality, known as cosmid SB12, were chosen for nucleotide sequence analysis.

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A. Construction of a cosmid library of DAS1529

Total DNA was isolated from DAS1529 with a DNA purification kit (Qiagen Inc., Valencia, CA). Vector and preparation, ligation, and packaging of the insert DNA following the instructions of the supplier (Stratagene, La Jolla, CA). DAS 1529 DNA inserts such as Sau 3A I DNA fragments were cloned into the Bam HI site of the SuperCos cosmid vector 1. The ligated product was packaged with the Gigapack® III gold packaging extract and transfected into host cells. XL1-Blue MRF '. The transformants were selected on LB-kanamycin agar plates. The cosmid library consisted of 960 randomly collected colonies that developed in ten 96-well microtiter plates in 200 µL of LB-kanamycin (50 µg / ml) for the selection of insect activity and long-term storage .

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B. Selection of the cosmid library of DAS1529 for evaluate insecticidal activity

For the primary selection of active clones against lepidopteran insects (CEW and TBW), a total of 960 cosmid clones were developed as individual colonies in 2 ml cultures in 96-well plates. The cultures were rotated and resuspended in original culture medium at a concentration of 10 X and subjected to bioassay. The SuperCos 1 vector (SB1) was included as a negative control. Sixteen positive clones (SB2 to SB 17) were isolated from the first round of selection. The second and third round of selection were carried out to select the activity against TBW and CEW; a cosmid clone (SB 12) that consistently showed the highest activity was chosen for further analysis. Table 5 summarizes the spectrum of activity (as tested) of cosmid SB12. (BAW the green beet donut, Spodoptera exigua; ECB is the European corn borer , Ostrinia nubilalis; SCRWis southern corn root worm, Diabrotica undecimpucata howardi .) The broth of the SB 12 E breeding ground. coli did not contain any activity against CEW; therefore, the active factors in SB12 were different from the active factors in the culture broth of the DAS strain
1529

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C. Sequencing of insertion of cosmid SB12 and identification of tc and cry type ORF

Nucleotide sequencing of cosmid SB12 showed that it contained a genomic insert of approximately 34 kb. Analysis of this sequence surprisingly revealed the presence of at least 10 assumed open reading frames (ORF) ( see Figure 2). Six of the ORFs identified were surprisingly found to have some degree of identity of amino acid sequences ( 38-48 %) to tcaA , tcaB , tcaC , and tccC identified above from Photorhabdus luminescens (Waterfield et al., 2001), Xenorhabdus nematophilus (Morgan et al., 2001), Serratia entomophila (Hurst and Glare, 2002; Hurst et al ., 2000), and Yersinia pestis (Cronin et al., 2001). The CT protein genes of Photorhabdus , Xenorhabdus , and Serratia have been shown to encode insecticidal factors. It was also very interesting that a DAS 1529 ORF had ~ 40% a Cry 1Ac amino acid sequence identity of Bacillus thuringiensis , another gene previously identified as an insecticidal factor (Schnepf et al., 1998; de Maagd et al ., 2001) . These findings had significant implication in the understanding of the distribution of toxin genes in the bacterial realm and in the development of additional strategies for genetically engineered extraction and modification of toxin genes.

The nucleotide sequence of the cosmid SB 12 was determined. The assembled DNA of 41,456 base pairs in length was further analyzed. Three holes were located two in the cosmid vector and one in the insert. Analysis of the nucleotide sequence of the longest contiguous approximately 34,000 base pairs revealed the presence of at least 10 assumed open reading frames (ORFs), identified as potential starting codons by extended open reading frames. This procedure is known to mistakenly identify translation starting sites in 19% ( Bacillus subtilis ) and 22% ( Bacillus halodurans ) in genomes related to Paenibacillus (Besemer, J., Lomsadze, A., Borodovsky, M ., Nucleic Acids Res. 29: 2607-2618,2001). Therefore, the quality and position of the complementary bases at the 5 'end of the 16S RNA of B. subtilis (review in Rocha, EPC, Danchin, A., Viari, A., Nucleic Acids Res. 27: 3567-3576 , 1999), N-terminal amino acid sequencing, and gene-related alignments were considered in the identification of native translation initiation sites. The assumed ORFs and annotations are summarized in Table 6 and are described in more detail at
continuation.

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ORF1 begins with the first nucleotide of the site of cloning for the DNA of DAS1529 in the funny SB12, and so So much is not your native translation start site. ORF1 shares significant DNA sequence homology with ORF3, and sequence comparison analysis suggests that the first 18 ORF1 base pairs are truncated, and that the first six codons encode amino acids Met-Val-Ser-Thr-Thr,  as found in OFR3. The beginning of the translation of ORF1 is presumably similar to that of ORF3, from approximately the bases 9505 to 9523 of SEQ ID NO: 1. Two predicted sequences of Amino acids are presented for ORF2, ORF4, and ORF6 (SEQ ID NOs: 19 and 13), based on translation start sites alternative, as indicated above. For ORF2, SEQ ID NO: 5 has been described above. The alternative startup site, and preferred is in the remainder 3295 of ORF1. In this way, the protein resulting from this start site would begin in the rest 9 of amino acids of SEQ ID NO: 5 (translation from RBS better). Of the Similarly, for ORF4, SEQ ID NO: 9 has been described above. He alternative start site, and preferred is in the rest 12,852 of SEQ ID NO: 1. The resulting protein would not have the first eight amino acids of SEQ ID NO: 9 (Starting this way with the amino acid residue 9 of SEQ ID NO: 8 - translation from RBS best).

Example 6

Sequence analysis of "duplicate" CTs

The degree of sequence identity was determined for the two fragments of ORF2 (tcaB1) compared to ORF4 (tcaB2), how was that for ORF1 (tcaA1) compared to ORF3 (tcaA2). Be observed a similar sequence relationship for both pairs of ORF.

ORF2 was constructed by combining two fragments, due to an insertion sequence type element that was inserted in nature (apparently spontaneously), and broke this ORF. See Figure 2. The location of this insertion can be determined by analyzing / comparing the entire DNA sequences of SB 12 (SEQ ID NO: 1) with the sequence of SEQ ID NO: 4, the last of which does not reflect (no code) the insertion. As indicated in parentheses in Figure 7, the 5 'translation product sequence before the remainder 490 of SEQ ID NO: 4 and the 3' translation product of the remainder 491 in alignment also with ORF4 (SEQ ID NO: 8). The DNA sequence at the apparent insertion point shows a direct repetition of 9 base pairs commonly found flanking the insertion elements (CACCGAGCA, bases 4734-4742 and 6071-6080 of SEQ ID NO: 1).

Example 7

Additional Sequence Analysis

In summary, according to the clustalW, GCG, and / or Blastp NTI vector analyzes, six of the identified ORFs (ORF1 to ORF6) had 38-48 % amino acid sequence identity with tcaA, tcaB, tcaC , and tccC (previously identified Photorhabdus tc genes). ORF7 encoded a protein that shared? 40% amino acid sequence identity with Cry 1Ac of Bacillus thuringiensis , another gene previously identified as an insecticidal factor. A phylogram was generated by incorporating the sequence of ORF7 ( Cry 1529) with a large number of Cry proteins (Figure 8). This phylogenetic tree suggests that Cry 1529 is distantly related to other P sequences. Cry popilliae such as Cry 18s (Zhang et al ., 1997, Zhang et al ., 1998) that are closer to Cry 2s; Cry 1529 decreases (remotely but not more closely) in a group of Cry proteins that commonly contain toxins found in lepidoptera ( Cry 1, Cry 9), beetles ( Cry 3, Cry 8, Cry 7), and dipterans ( Cry 4 ), which is a different group compared to those that include Cry 5, -12, -13, -14, and -21 and Cry 2, -18 nematode toxins.

It was a surprising and novel discovery to find the Cry and CT protein genes (in the genomic insertion of SB12) in Paenibacillus . The identification of new Cry and TC protein genes has relevance with the understanding of the technique of Photorhabdus and Xenorhabdus, and Bacillus thuringiensis , and broadens the scope of the bacterial genus in which the Cry and TC protein genes have been found. The full length Cry 1529 size identified herein corresponds to the Cry 1s core toxin; Cry 1529 represents a new class of Cry proteins that also have implications for isolating cry genes from Bacillus thuringiensis and Paenibacillus.

To verify that these surprising observations were not the result of strain contamination ( i.e. to confirm that the 34 kb insertion that carries CT and Cry ORF was in fact DAS1529 DNA), molecular analysis was carried out by hybridization. Southern blotting and PCR. For PCR validation, specific primers of ORF6 ( tccC type) and ORF7 ( Cry 1529) (Example 8, Table 8) were used to amplify ORF6 and ORF7 from cosmid SB 12 and total DNA of DAS 1529. For ORF6, performed PCR amplifications on a PE9600 thermal cycler (Perkin Elmer) with the following parameters: initial denaturation at 95 ° C for 2 minutes; 30 cycles each with denaturation at 95 ° C for 30 seconds, hybridization at 60 ° C for 45 seconds, extension at 72 ° C for 2 minutes, and a final extension for 10 minutes at 72 ° C. For ORF7, the amplification parameters were the same as ORF6, except that the hybridization temperature was 55 ° C for 30 seconds and extension at 72 ° C for 4 minutes. Specific PCR products with a single band of expected sizes were amplified for both ORF6 and ORF7.

Initial southern blot hybridization was based on a partial DNA sequence of SB 12 DNA and was carried out according to the standard protocol (Sambrock et al., 1990). DNA samples included total DAS1529 DNA from two independent preparations, cosmid SB 12 DNA, and a NC1 negative control DNA sample ( Photorhabdus ). Both DNA samples from DAS1529 were 16S rDNA confirmed that it was of Paenibacillus sp . Origin, and one was originally used for the construction of the cosmid library; The other was a new preparation. DNA samples were digested with Eco RI , transferred to membrane, and hybridized with 180 labeled base pairs of the amplified PCR product outside of SB 12 Roche DIG System (Roche). The PCR primers are 5 'CCT CAC TAA AGG GAT CAC ACG G 3' that partially hybridized with the vector and truncated ORF1 (compared to full length ORF3), and 5 'GGC TAA TTG ATG AAT CTC CTT CGC 3' which hybridizes to truncated ORF1 ( tcaA type) and full length ORF3 ( tcaA type). A total of three DNA fragments (0.85, 2.7, and 8.0 kb) that hybridized to the PCR probe were detected, 0.85 and 8.0 in SB12 and 2.7 and 8.0 in the DNA of DAS 1529. No signal was detected in the negative control. The 0.85 kb (from the internal Eco RI fragment ORF1 to the first Eco RI site in the vector) and 8.0 kb (from the first 5 ' Eco RI site in ORF3 to the third EcoRI site in ORF1) matched the sizes calculated from the target DNA fragments of SB12. Detection of the 2.7 kb fragment fragment suggests the presence of a 2.7 kb Eco RI site immediately upstream of the Eco RI site within ORF1 in the DNA of DAS 1529. Th. The results show that the insertion of SB12 was from the total DNA of DAS 1529 and, based on hybridization and restriction analysis, all copies of the ORFs were responsible for it.

Example 8

Characterization of protein insecticidal activities encoded by cosmid ORFs

Random transposon insertion mutagenesis (to disrupt the individual ORF or an entire operon) and heterologous expression (expressing the individual ORFs or complete operons) were addressed to isolate the ORF (s).
individual (s) or operons that confer insecticidal activities on the cosmid SB 12.

A. Mutagenesis of random transposons of the cosmid SB12

Tn mutagenesis was generated from cosmid SB12 of DAS1529 A Tn using the GPS-1 Genome Priming System (New England BioLabs, Beverly, MA) following the kit instructions. In summary, 2 µl of 10 X GPS buffer, 1 µl of DNA donor from pGPS2.1 (0.02 µg), 1 µm of cosmid SB12 (0.1 µg) and 18 µl of H_ {2 } Or sterile were added to a 0.5 ml tube. One ml of TnsABC Transposase was added; The mixture was stirred in a Vortex apparatus and spun briefly to collect the materials at the bottom of the tube. This reaction mixture was incubated for 10 minutes at 37 ° C. One µl of starting solution was added and mixed by pipetting up and down several times. The reaction was incubated at 37 ° C for 1 hour and then quenched at 75 ° C for 10 minutes. One µl of the reaction mixture was diluted 10 times with sterile H2O; 1 µl of the diluted reaction was electrophoresed in 100 µL of Electro MAX DH5α-E E. coli (Gibco BRL, Rockville, MD). After 1 hour of growth in SOC medium at 37 ° C, 10 µl or 100 µl were seeded on LB agar plates containing 20 µg / ml Kanamycin and 15 µg / ml chloramphenicol, followed by incubation for overnight at 37 ° C.

Individual colonies of the T12 mutagenesis of SB12 in recent LB agar plates that contained 20 µg / ml Kanamycin and 15 µg / ml of chloramphenicol From stretch marks, 50 ml cultures of LB that it contained 20 µg / ml Kanamycin and 15 µg / ml chloramphenicol was inoculated and developed at 28 ° C, 200 rpm for 48 hours. The cells were then collected by centrifugation at 3500 rpm during 20 minutes. The supernatant was removed and returned to suspend the sediment in 2.5 ml of the culture supernatant to a concentration of 20 X. The concentrated cell pellet is then tested to determine the activity against the worm of the corn. Forty \ mul of the surface were applied 20 X concentrate on artificial diet using 8 wells per sample in 128-well plates. Worm larvae were added Freshly incubated corn cob and allowed to feed for 5 days, at which time the mortality was recorded and pesos.

A total of 184 clones were tested to determine the loss of activity against the corn cob worm. The results are summarized in Table 7. Bioassays of the Tn clones revealed that an insertion of Tn into the Cry 1529 gene that results in the complete loss of activity. The initial bioassay showed that the activities of the clones that carried the Tn insertions in the tc genes were variable. Subsequent analyzes of the clones in which the cultures were all normalized to the same cell density before the bioassay did not show any loss of activity when compared to SB12. The results of the Tn analysis suggest ORF7 ( Cry 1529) is the key insecticidal active component of the cosmid SB 12.

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B. Heterologous expression of ORF / operon of SB12

Cry 1529 (ORF7) and five tc ORFs ( see Table 8 below) were expressed in the pET101D® system. See Figure 5. This expression vector has all the attributes of the T7 regulated pET expression system (Dubendorff and Studier, 1991; Studier and Moffatt, 1986) and allows the directional cloning of a blind end PCR product into a vector for high level regulated expression, and simplified protein purification in E. coli Optimal PCR amplification employed PfuTurbo ? Fidelity high- fidelity DNA polymerase that is highly thermostable and has a 3-to-5 'exonuclease reading-proof activity to correct errors of incorrect nucleotide incorporation (Stratagene , La Jolla, CA). When ThermalAce? Polymerase (Invitrogen) is used, point mutations were introduced into the tc ORFs, which were corrected by the site-directed Quick-Change? XL mutagenesis kit based on PfuTurbo ? ( Stratagene) E. coli strain BL21 Star? (DE3), was used as a host for expression as it contains the rne131 mutation (López et al., 1999) which generally enhances mRNA stability and production of recombinant proteins

The individual ORFs were amplified by PCR outside the cosmid SB12 with specific ORF primers (Table 8) under defined conditions. As a requirement of directional cloning, the direct PCR primers were designed to contain the sequence, CACC, at the 5 'end to secure the base pair of the PCR product with the outgoing sequence, GTGG, in vector pET101 .D. Inverse primers when paired with direct primers will amplify each ORF, respectively. PCR reactions were carried out in 50 µl of reaction mixture containing 50 ng of the cosmid SB12 DNA, 1 X of Pfu reaction buffer (Stratagene), 0.2 mM each of dNPT, 0.25 mM of each primer, and 2 U of PfuTurbo (Stratagene) DNA polymerase. Amplifications by PCR were performed on a PE9600 thermal cycler (Perkin Elmer) with the following parameters: initial denaturation at 95 ° C for 2 minutes, 35 cycles each with denaturation at 95 ° C for 30 seconds, hybridization at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes per kb of ORF, and a final extension for 10 minutes at 72 ° C.

TABLE 8 Summary of PCR primers for cloning from ORF1-7

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The PCR products for each ORF were cloned in pET101.D following the instructions of the supplier (Invitrogen). The cloned ORF was purified as plasmid DNA from pET101.D and was He verified the sequence. Since the Tn analysis indicated that the ORF7 It is the key component of SB 12 for pest control tested, the biochemical analysis and the insect bioassay were focused on heterologous ORF7 proteins. For ORF7 expression clones, sequence analysis of DNA showed 100% concordance with the DNA sequence of SB12 original. The expression of ORF7 was induced by 0.5 mM IPTG for 4 hours according to kit instructions (Invitrogen).

C. Bioassay to determine insecticidal activities of ORF7 and operon tc

Bioassay samples were prepared whole E. coli cells, cell lysates, and purified toxins. The specific spectrum and activity of ORF7 ( Cry 1529) is summarized in Table 10. Cry 1529 is more active against the horned tobacco worm ( Manduca sixth ) and highly active (LC50 of 10 µg / ml diet) against the tobacco bud worm ( Heliothis virescens ); 100% mortality was observed for both insects. At higher doses, Cry 1529 conferred some mortality (20 to 60%) and inhibition of substantial growth on the corn cob worm ( Heliothis zea ), green beet donut ( Spodoptera exigua ), and black cut worm ( Agrotis ipsilon ). For the European corn borer ( Ostrinia nubilalis ), Cry 1529 had some growth inhibition at higher doses. For some other insect species (autumn black caterpillar, cricket of the cotton capsule, southern root worm, mosquito), it did not detect any activity. The values of CL 50 of Cry 1529 for colonies of the black diamond resistant moth (DBMr) and black diamond moth sensitive (DBM) to Cry 1A ( Cry 1Ac) and are> 50 µg / ml and <1.0 µg / ml, respectively, suggesting a potential cross resistance. Cry 1529 did not confer detectable activity on grass weevils, a relative of Japanese beetles.

To test the activity of other non- Cry 1529 factors in DAS 1529, an inactivated C12 cosmid clone of Cry 1529 tn (tn67) was tested against TBW, CEW, SCRW, ECB, BW, BAW, THW, and grass weevils ; No activity was found against these pests. To direct the distribution of non-expression or low expression of tc ORF in the formation of SB 12, tc ORF expressed in a manner and in combination with other CTs of DAS 1529 were independently tested; No activity was found against TBW, CEW, and grass weevils. In addition, four ORFs were expressed in the form of a single operon up to very high levels in E cells. coli When tested in vitro , whole cells did not contain any detectable activity on TBW, CEW, and grass weevils. Although the lack of weevil activity is somewhat interesting, these results are not surprising because Paenibacillus typically infects a narrow host range of weevils, it could follow that the spectrum of activity of insecticidal toxins could be relatively narrow. Thus, selections (using known procedures) that imply a wider range of pests, and additional time, would be required to identify susceptible pests. The results presented herein should not lead anyone out of recognizing that the subject CT proteins are useful as do other TC proteins of Xenorhabdus , Photorhabdus , and the like.

Soluble proteins were extracted with 25 mM sodium phosphate pH 8.0, 100 mM sodium chloride and analyzed on 4-12% NuPAGE gradient gel with 1 X MES buffer (Invitrogen). The ORF7 protein was purified using conventional procedures and N-terminal sequencing revealed the expected sequence: MNSNEPNLSDV. A bioassay was performed with whole E cells. coli , with normalized cell density, expressing target proteins. See Figure 6. The large-scale purified ORF7 protein was used to obtain the LC50 values for ORF7 by in vitro bioassay. The thermal stability analysis of the purified ORF7 indicated that a 5 minute treatment at 75 ° C is sufficient to abolish its activity against TBW. See Table 9.

TABLE 9 Thermal stability of purified Cry 1529 (ORF7)

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For the tc genes, error-free ORF3 and ORF6 clones were used as intermediate clones to generate tc operon clone expressing ORF3 ( tcaA ), ORF4 ( tcaB ), ORF5 ( tcaC ), and ORF6 ( tccC ). To construct the tc operon in pET101.D, Nsi I / Sac I fragment containing partial TCAA, Tcab integer and TCAC, and cleaved partial TCCC out cosmid SB 12 to replace the insert Nsi I / Sac I in pET101.D -tcaA; This was followed by the insertion of a fragment of 208 Sac I base pairs of pET101.D-tccC. See Figure 5. The four ORFs were expressed at high levels by the standard introduction of IPTG. For the ORF6 (tccC) expressed in the tc operon, the size of the expressed protein was significantly smaller than the ORF6 predicted by the NTI vector of the 5'-most ATG (SEQ ID NO: 18) and expressed in a manner Independent. Therefore, the annotated ORF6 (SEQ ID NO: 13) based on the presence of a ribosome that binds to the consensus site is probably the native protein produced in SB 12 and
DAS1529.

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D. Toxin Spectra Activity

The spectrum of activity of Cry 1529 toxin (ORF7) is summarized in Table 10.

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Only a limited range of pests is used in trials in an initial attempt to determine the spectrum of ORF activity of TCs / tc subject. The following were obtained data, which use the ORF3-OR6 operon:

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Again, although this initial round of selection did not reveal the activity of these pests, those skilled in the art will not doubt that the subject proteins are useful, as are the corresponding Photorhabdus / Xenorhabdus proteins. In addition, see Example 10, below.

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Example 9

Use of PCR primers to identify homologues of Cry 1529 from other genera, species, and bacterial strains

For additional selection homologs of cry 1529 of ORF7 from other strains ( Paenibacillus or others), gene specific and degenerate PCR primers were designed to amplify the 1 kb target ORF7 DNA sequences. PCR primers were derived from well-conserved protein motifs (QAANLHL, domain I, block 1 core for the master primer; GPGFTGGD, domain III, block 3 for the reverse primer) highly conserved in Cry proteins. The primers are listed in Table 12 and validated on DAS 1529. The PCR amplifications were performed on a PE9600 thermal cycler (Perkin Elmer) with the following parameters: initial denaturation at 95 ° C for 2 minutes; 35 cycles each with denaturation at 95 ° C for 30 seconds, hybridization at 47 ° C for 45 seconds, extension at 72 ° C for 2 minutes, and a final extension for 10 minutes at 72 ° C. Primer pairs were used to select a bacterial culture collection (not B. thuringiensis ) by PCR. Five of 192 strains (three Paenibacillus, one Bacillus , and one unidentified) produced PCR products of expected sizes. These strains were also found to have CEW activity according to a primary bioassay selection. However, sequence analysis of the amplicons obtained from one of these strains. However, sequence analysis of the amplicons obtained from one of these strains using different primers showed that the amplicons were not derived from a cry gene.

Despite this, and since these selections were not exhaustive, the present invention includes methods for selecting Paenibacillus spp., Bacillus spp. ( including Bacillus thuringiensis and sphaericus ), and the like for Cry 1529 type genes and proteins. Given the significant nature of the discovery of toxic Cry proteins for lepidopterans - in Paenibacillus, the present invention also includes methods for selecting Paenibacillus spp., in general, for toxic Cry proteins and genes for lepidoptera. Various selection procedures are known in the art, including PCR techniques (as exemplified above), probes and feeding assays (where whole cells are fed to the target pests). Those skilled in the art will readily recognize, selection methods of the present invention include the preparation and use of clone libraries (such as cosmid libraries) in these selections.

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Example 10

Complementation of the Xenorhabdus XptA2 TC TC protein toxin with DAS1529 TC proteins

This example provides experimental evidence of the ability of DAS 1529TC proteins, expressed here with a single operon (ORF 3-6; tcaA, tcaB, TcaC and tccC; see section C of Example 8), to complement the XptA2 toxin of Xenorhabdus nematophilus Xwi ( see SEQ ID NO: 49). Two independent experiments were performed to express the CT operon of DAS 1529 and XptA2 independently, or to co-express the XptA2 gene and the CT operon in the same E. coli cells. Whole cells expressing different toxins / toxin combinations were tested to determine activity against lepidoptera insects: corn cob worm ( Heliothis zea; CEW ) and tobacco bud worm ( Heliothis virescens; TBW ). Data from both experiments indicate that the DAS 1529 TC proteins are capable of enhancing the XptA2 activity of the Xenorhabdus CT protein on both insect species tested.

A. Co-expression of the CT scans of DAS1529 and Xenorhabdus XptA2

Expression of the CT operon was regulated by the T7 promoter / lac operator in the expression vector pET101.D which carries an origin of ColE1 replication and an ampicillin resistance selection marker (Invitrogen). The exhaustive description of the cloning and expression of the tc operon can be found in section C of Example 8. The XptA2 gene was cloned into the expression vector pCot-3, which carries a chloramphenicol resistance selection marker and an origin of Replication compatible with ColE1. The expression system of the pCot-3 vector is also regulated by the T7 promoter / lac operator. Therefore, compatible origins of replication and different selection markers form the basis for the co-expression of the CT and XptA2 operon in the same E. coli cells. Plasmid DNAs carrying the CT operon and XptA2 were transformed into E. coli, BL21 Star ™ (DE3) either independently or in combination. The transformants were selected on LB agar plates containing 50 mg / ml carbenicillin for the pET101.D-TC operon, 50 mg / ml chloramphenicol for pCot-3-XptA2, and both antibiotics for the pET101.D-TC operon / pCot-3-XptA2. To suppress basal toxin expression, glucose was included at a final concentration of 50 mM in both agar and LB liquid medium.

For the production of toxin, 5 ml and 50 ml of LB medium containing antibiotics and 50 mM glucose were inoculated with overnight cultures that were grown on LB agar plates. The cultures were grown at 30 ° C on a shaker at 300 rpm. Once the culture density has reached a D.O of ~ 0.4 at 600 nm, IPTG at a final concentration of 75 mM was added to the culture medium to induce gene expression. After 24 hours, E. coli cells were collected for protein gel analysis by the NuPAGE system (Invitrogen). The sediments of 0.5 ml cells of 1 X culture broth were resuspended in 100 ml of 1 X NuPAGE LDS sample buffer. After a brief sonication and boiling for 5 min, 5 µl of the sample was loaded into 4 to 12% gradient NuPAGE bistro gel for total total protein profile analysis. XptA2 expressed at detectable levels when expressed independently or in the presence of the CT operon. Based on the gel scanning analysis using a Personal Densitometer SI (Molecular Dynamics), XptA2 expressed approximately 8 X as high by itself as when it is co-expressed with the CT operon. For the 5 ml induction experiment, there is an almost equal expression of XptA2.

B. Bioassay for Insecticidal Activity

As described in Example 8, the tc ORFs of DAS 1529, when expressed independently or as an operon, did not appear to be active against TBW and CEW. The following bioassay experiments focused on determining whether Paenibacillus CT proteins (DAS1529) (from ORFs 3-6; TcaA-, TcaB-, TcaC-, and TccC- type proteins) can complement the activity of the protein toxin Xenorhabdus TC TC (XptA2 is exemplified). Bioassay samples were prepared as whole E cells. coli in 4 X of cell concentrate for the 5 ml induction experiment, both the XptA2 and XptA2 / TC operon cells contained very low but almost equal amount of the XptA2 toxin. The data in Table 13 showed that at the concentration of 4 X cells tested, TC + Xenorhabdus XptA2 proteins were active against CEW. This provided the first evidence of a complementing effect of Paenibacillus DAS 1529 TC proteins on Xenorhabdus XptA2 .

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TABLE 13 Xeno DAS1529 CT complementation bioassay. XptA2 on H. Zea

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For the second bioassay experiment, the amount of XptA2 protein in the XptA2 cells and the XptA2 + CT operon cells were normalized based on the densitometer gel scan analysis. As shown in Table 14, XptA2 per se had a moderate activity at 40 X over TBW ( H. Virescens ), but the activity fell to an undetectable level a and below 20 X. However, when co-expressed with CT scans were very evident high levels of activity in the presence of 10 X and 5 X of XptA2, and low activity at 1.25 X of XptA2 was still detectable. These observations indicate that there is a significant potentiation effect of the 1529 CT proteins on Xenorhabdus XptA2 against H. virescens At the highest doses tested, neither the negative control nor the tc operon per se had an activity against this pest.

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TABLE 14 XptA2 IDAS1529 CT complementation bioassay on H. virescens

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Example eleven

Stabilization of the Cry 1529 protein against trypsin digestion

This example teaches for modifications of the DNA sequence described as SEQ ID NO: 14, which encodes the Cry 1529 protein (described as SEQ ID NO: 15) so that the new encoded proteins are more resistant to proteolytic digestion for trypsin which is the native protein. Protein digestion in the insect intestine limits the exposure time of the insect to a protein toxin. Therefore, methods can be used to decrease the susceptibility of a protein toxin to protease digestion to increase the potency of the protein.

For these tests, the trypsin enzyme (for example, Sigma Chemical No. T1426) and trypsin inhibitors ( for example, Sigma Chemical No. T9008) were prepared as stock solutions of 4 mg / ml or 10 mg / ml in 50 mM Tris HCl buffer , pH 8.0. Assay incubations with various concentrations of trypsin and Cry 1529 protein were performed at 37 ° C for 1 hour, and were terminated by the addition of an equal volume of an equal concentration of trypsin inhibitors ( eg, a digestion that received 35 ml of 4 mg / ml trypsin solution was terminated by adding 35 ml of 4 mg / ml trypsin inhibitors). For a typical experiment, the Cry 1529 protein was produced by appropriately engineered modification of E cells. coli and was purified by the steps described above, which included the separation of other proteins by passage through a size exclusion column. After digestion, protease products were analyzed by standard archilamide gel electrophoresis followed by immunoblot analysis using antibody prepared against Cry 1529. The results of such an experiment are shown in Figure 9.

Trypsin digestion produces two main protein products, the smallest being approximately 50 kDa in molecular size. It is indicated that this digestion pattern is the same as that produced from trypsin digestion of a Cry 1529-His6 protein, which is identical to the amino acid sequence of the native Cry 1529 protein SEQ ID NO: 15 except for the amino acid addition

KGELNSKLE GKPIPNPLLGLDSTRTGHHHHHH to the carboxy end. The coding region for Cry 1529-His6 was produced by ligands the coding region for the native Cry 1529 protein in vector pET101 / D-TOPO® (Invitrogen ™, Carlsbad, CA). This recombinant clone was performed to facilitate the purification of the Cry 1529 recombinant protein by binding to a commercially available V5 antibody, whose epitopes are represented by the amino acid sequence GKPIPNPLLGLDSTRTG (underlined above), or by purification schemes using the six histidine residues (double underlined above). The procedures for these manipulations were performed in accordance with the recommendations provided with the vector pET101 /
DTOPO®.

Trypsin digestion of the Cry 1529-His6 protein was found to eliminate activity in insect bioassays against lepidopteran insects. MALDI-TOF analysis was used to determine the sequence of amino acids that make up the N-terminus of the 50 kDa peptides, and two protease processing sites were determined corresponding to amino acid residues 122 (R, Arginine) and 126 (K, Lysine) of SEQ ID NO: 15.

Modifications the first cleavage site of trypsin in the encoded protein were performed in the sequence DNA native (SEQ ID NO: 14), using the mutagenesis methodology from Quick-Change® (Stratagene, La Jolla, CA). Three different types of mutations were made in amino acids in the region 120 to 123 of SEQ ID NO: 15: RARE to HANA, RARE to RARS, and RARE to QANA. DNA oligonucleotide primers (listed in  5 'to 3' address for each strand) for these mutations are listed in Table 15 below. The bases that differ from The native DNA sequence are underlined.

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TABLE 15

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Comparison of coding regions of wild type and mutates induced by these primers are shown in this table. Relevant amino acid residues are shown in bold font

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The separate, mutated coding regions were each cloned into the vector pET101 / D-TOPO®, which allows inducible production of the Cry 1529 variant proteins. E. coli cells containing the constructs were developed, and the expression of Cry 1529 variant genes by procedures recommended by the provider. The whole cells collected were then tested in trypsin digestion assays, and analyzed as before. Typical results are shown in Figure 10. For these experiments, 10 mg of whole cell sediment was suspended in 50 mM Tris HCl, pH 8.0, and digested for 3 hours at 37 ° in a final volume of 1 ml, with 100 ml of 10 mg / ml trypsin. The reactions were mixed occasionally during incubation. Digestion was terminated by the addition of 100 µL of 10 mg / ml trypsin inhibitors and the tubes were stored in tubes.

These results demonstrate that both Cry 1529 (RARA) and native Cry 1529-His 6 (RARA) proteins were digested by trypsin to produce a main product of approximately 50 kDa. When the corresponding RARA sequence the trypsin cleavage site was mutated to HANA or QANA, substantial resistance to trypsin digestion was obtained. No 50 kDa peptides were produced, and readily detectable amounts of apparently full-length Cry 1529-His 6 proteins were present. The RARA site mutation for RARS did not eliminate the production of 50 kDa peptides, but substantially reduced the protease cleavage rate. Thus, it is evident that the protease processing sites in the Cry 1529 protein substantially decreases its susceptibility to protease digestion. This allows proteins to reside longer periods of time in the insect intestine after ingestion, resulting in an increase in potency to eliminate susceptible insects.

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Example 12

Design PCR primers for the detection of homologs of IDAS 1529 tcORFs in other strains of Paenibacillus

As shown above, strain IDAS 1529 of Paenibacillus produces an extracellular protein that is toxic to various Lepidopteran insects. The molecular phylogeny of the 16S ribosomal gene of this strain indicates that it is most closely related to the members of the P. thiaminolyticus-P group . lentimorbus-P. Popilliae It has also been shown that Paenibacillus IDAS 1529 strain has both the toxin complex genes (hereinafter referred to as tc genes) and a novel insecticidal crystalline inclusion protein gene called cry 1529. In another attempt to determine whether Tc homologues are present members of the Paenibacillus genus, a collection of Paenibacillus strains was selected by polymerase chain reaction (PCR) and hybridization analysis. For PCR analyzes, total DNA isolated from Paenibacillus strains was used as a template and selected using oligonucleotide primers specific for tc genes found in IDAS strain 1529 of Paenibacillus , Photorhabdus , and Xenorhabdus species. The amplified products obtained with the tc primer sets were cloned and their nucleotide sequence was determined and compared with the tc sequences obtained from Paenibacillus strain IDAS 1529 . The following examples illustrate how tc- specific oligonucleotides and PCR use can be designed for the investigation of total DNA from Paenibacillus isolates for DNA sequences that are homologous to the tc genes identified in IDAS strain 1529 of Paenibacillus species, Photorhabdus , and Xenorhabdus species. Using the PCR analysis (as described herein), it was (and is) can identify homologs tc in a species of Paenibacillus different strain IDAS 1529 Paenibacillus and P. thiaminolyticus-P group . lentimorbus- P. popilliae .

12.A. - Total DNA extraction from Paenibacillus strains

Paenibacillus strains on nutrient agar plates (8 g / l nutrient broth, 15 g / l Bacto agar; Difco Laboratories, Detroit, MI) for 3-5 days at 30 ° C. A single colony was collected and inoculated in a 500 ml flask with three baffles containing 100 ml of sterile nutrient broth (8 g / l of nutrient broth; Difco Laboratories, Detroit, MI). After 24-72 hours of incubation at 30 ° C on a rotary shaker at 150 rpm, the cultures were dispensed in sterile 500 ml polyethylene bottles and centrifuged at 6,500 xg for 1 hr at 4 ° C. After centrifugation, the supernatant fluid was decanted and the bacterial cell pellet was retained. Total DNA was extracted from sediment from cells using the QIAGEN Genomic-tip 100 / G system and Geonomic DNA buffer set (QIAGEN Inc., Valencia, CA, United States) following the Sample and Lysis Protocol for Bacteria exactly as described by the manufacturer. Total extracted DNA was solubilized in 0.5 ml of TE buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA, pH 8.0).

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12.B. - Selection of tc oligonucleotide primers for PCR

To select the specific oligonucleotide primers of the tc genes previously identified from Paenibacillus strain IDAS 1529, the nucleotide sequences of tcaA, tcaB, tcdB and tccC obtained from Paenibacillus strain IDAS 1529, strain W14 of Photorhabdus , and Xenorhabdus strain Xwi were aligned using the Align program in the Vector NTI software package (Informax, Inc., Frederick, MD). The nucleotide sequences used for this analysis are listed in Table 17.

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12.Bi - TcaA specific primer selection

The nucleotide sequence alignment of tcaA1-1529, tcaA2-1529 , and tcaA-W14 identified two nucleotide sequence identity regions of sufficient length for the selection of PCR primers with minimal degeneration (shown as boxed regions in Figure 10.). These two regions were selected for the synthesis of tcaA specific primers, which were designated SB 105 and SB 106 (Tables 18 and 19).

12.B.ii. - Selection of tcaB specific primer

The nucleotide sequence alignment of tcaB1-1529, tcaB2-1529 , and tcaB-W14 identified four nucleotide sequence identity regions of sufficient length for the selection of PCR primers with minimal degeneration (Figure 11.). These four regions were selected for the synthesis of tcaB specific primers, which were designated SB101, SB102, SB 103, and SB104 (Tables 18 and 19).

12.B.iii. - Selection of tcaC specific primer

The alignment of nucleotide sequences of tcdB1-W14, tcdB2-W14, xptC1-Xwi and tcaC-1529 identified two nucleotide sequence identity regions of sufficient length for the selection of PCR primers with minimal degeneration (Figure 12 .). These two regions were selected for the synthesis of tcaC specific primers , which were designated SB215 and SB217 (Tables 18 and 19).

12.B.iv. - Selection of specific tccC primer

The nucleotide sequence alignment of tccC1-W14, tccC2-W14, tccC3-W14, tccC4-W14, tccC5-W14, xptB1-Xwi and tccC-1529 identified two nucleotide sequence identity regions of sufficient length for the selection of PCR primers with minimal degeneration (Figure 13.). These two regions were selected for the synthesis of specific tccC primers, which were designated SB212 and SB213 (Tables 18 and 19).

TABLE 18 Tc - specific primers

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TABLE 19 Tc primer combinations

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Example 13

PCR amplification of Paenibacillus DNA

For PCR amplification using sets of tcaA- and tcaB - specific primers, 3-5 ul of total DNA from each of the Paenibacillus strains were mixed with 50 pmoles of each primer and 1 X Eppendorf MasterMix (Eppendorf AG; Hamburg, Germany) in a reaction volume of 20 ul. The amplification conditions were denatured at 94 ° C for 3 minutes followed by 30 cycles of denatured at 94 ° C for 1 minute, hybridization at 52 ° C for 1.5 minutes, and extension at 72 ° C for 1.5 minutes, followed by a final extension at 72 ° C for 5 minutes.

For PCR amplification using sets of specific tcaC- and tccC primers , approximately 375 ng of total DNA obtained from each of the Paenibacillus strains was mixed with 50 pmoles of each primer and 12.5 ul of Epicenter buffer ® FailSafe ™ Dand 2.5 U of Epicenter® FailSafe ™ Polymerase (Epicenter; Madison, WI) in a reaction volume of 25 ul. The amplification conditions will be denaturation at 96 ° C for 4 minutes followed by 40 cycles of denaturation at 94 ° C for 30 seconds, hybridization at 64 ° C for 30 seconds, and extension at 70 ° C for 30 seconds. Each cycle, the hybridization temperature was reduced by 0.5 ° C and the extension time increased by 5 seconds.

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13. A. - Gel electrophoresis, cloning, and product nucleotide sequence determination PCR amplified

PCR amplification reactions were examined by gel electrophoresis using 0.8 to 1% Seakem LE agarose (BioWhittaker Molecular Applications, Rockland, ME) in 1 X TAE buffer. The amplified products were cloned into the pCR 2.1-TOPO® vector using the TOPO TA® Cloning Kit (Invitrogen ™ Life Technologies, Carlsbad, CA) exactly as described by the manufacturer. The nucleotide sequences of the total amplified products were determined using direct M13, reverse M13, and tc sequence specific sequencing primers as needed to obtain the double chain sequence of each amplified product. Nucleotide sequencing was performed using the CEQ Dye Terminator Cycle Sequencing Quick Start kit (Beckman Coulter, Fullerton, CA, United States) and the CEQ 2000 XL DNA Analysis System (Beckman Coulter) exactly as described by the manufacturer. The Sequencher software package (v. 4.1.4) (Gene Codes, Ann Arbor, MI) was used to construct contiguous sequencing data and determine a consensus sequence for each amplified product.

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13. B. - Nucleotide sequence analysis of products amplified by PCR 13.Bi - tcaA

When PCR was used, tcaA- (combination of primer SB105 and SB106) was performed using total DNA obtained from the collection of Paenibacillus strains, it was observed that the total DNA of a strain of Paenibacillus apiarius (NRRL NRS 1438; de hereinafter referred to as DB482) produced an amplified product of the expected sizes. The amplified product was cloned and sequenced.

The amplified product obtained using the combination of primers of SB105 and SB106 was called tcaA2-DB482. When the tcaA2-DB482 sequence (SEQ ID NO: 32) is compared with the tcaA sequences obtained from the Paenibacillus strain 1529 and Photorhabdus strain W14, it was observed that tcaA2-DB482 had the highest nucleotide sequence identity (90 , 5% on 1,239 nucleotides) to tcaA2-1529 (Table 20). The deduced amino acid sequence encoded by tcaA2-DB482 (called TcaA2-DB482; SEQ ID NO: 33) would be 89.1% identical to the corresponding amino acid sequence of tcaA2-1529 (called TcaA2-1529; SEQ ID
NO: 7).

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3. 4

13.B.ii. - tcaB

The amplified products obtained using the combination of primers of SB 101 and SB 102 and the combination of primers of SB103 and SB104 are called tcaB2a-DB482 and tcaB2b-DB482 , respectively. When the sequences of tcaB2a-DB482 (SEQ ID NO: 34) and tcaB2b-DB482 (SEQ ID NO: 35) were compared with the sequences of tcaB obtained from strain IDAS 1529 of Paenibacillus and strain W14 of Photorhabdus , it was observed that both sequences had the highest identity of nucleotide sequences with tcaB1-1529 and tcaB2-1529 (Table 21). The nucleotide sequence identity of tcaB2a-DB482 and tcaB2b-DB482 to tcaB2-1529 was 92.6% and 89.8%, respectively. The deduced amino acid sequences encoded by tcaB2a-DB482 (called TcaB2a-DB482; SEQ ID NO: 36) tcaB2b-DB482 (called TcaB2b-DB482; SEQ ID NO: 37) were 91.2% and 91.1% identical, respectively , with the corresponding deduced amino acid sequence of tcaB2-1529 (named TcaB2-1529; SEQ ID NO: 9).

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13. B.iii. - tcdB

When the PCR using the combination of tcaC specific primers (SB215 and SB217) was performed using the total DNA obtained from DB482 an amplified product of the expected size was produced. The amplified product was cloned and sequenced.

The amplified product obtained using the combination of primers of SB215 and SB217 was called
tcaCDB482. When the tcaC-DB482 sequence (SEQ ID NO: 38) was compared with the tcaC sequences obtained from strain IDAS 1529 of Paenibacillus , strain Xwi of Xenorhabdus and strain W14 of Photorhabdus , it was observed that tcaCDB482 had the identity of Major nucleotide sequences (93.5% over 2,091 nucleotides) at tcaC-1529 (Table 22). The deduced amino acid sequence encoded by tcaC-DB482 (called TcaC-DB482; SEQ ID NO: 39) was 91.1% identical to the corresponding amino acid sequence deduced from tcaC-1529 (named TcaC-1529; SEQ ID NO: 11 ).

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13. B.iv. - tccC

When the PCR using the combination of specific tccC primers (SB212 and SB212) was performed using the total DNA obtained from the Paenibacillus strain collection, it was observed that the total DNA of DB482 produced an amplified product of the expected size. The amplified product was cloned and sequenced.

The amplified product obtained using the combination of primers of SB212 and SB213 was called tccC-DB482. When the tccC-DB482 sequence (SEQ ID NO: 40) was compared with the tccC sequences obtained from strain IDAS 1529 of Paenibacillus , strain Xwi of Xenorhabdus and strain W14 of Photorhabdus , it was found that tccCDB482 had the identity of Major nucleotide sequences (93.7% over 858 nucleotides) at tccC-1529 (Table 23). The deduced amino acid sequence encoded by tccC-DB482 (called TccC-DB482; SEQ ID NO: 41) was 95.5% identical to the corresponding amino acid sequence deduced from tccC-1529 (named TccC-1529; SEQ ID NO: 13 ).

TABLE 23 Nucleotide amino acid sequence identity and deduced from the corresponding regions of tccC-DB482 of xptB1 - Xwi , tc-W14 , tccC-1529 , and tcc genes of Photorhabdus strain W14

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13.C. - Summary of PCR analyzes

This example (and other examples herein) illustrate procedures for designing oligonucleotide primers based on the tc genes of the three bacterial genera, and that the use of these primers for PCR selection of Paenibacillus strains can identify homologues of tc present in those strains. DB482, which is an isolation of Paenibacillus apiarius (deposited as NRRL B-30670) that was isolated from honey bee larvae, was shown to contain homologues of tcaA, tcaB, tcaC , and tccC . The finding of these tc homologues confirms that the IDAS 1529 strain of Paenibacillus is not unique within the Paenibacillus genus in relation to the possession of the tc genes. Therefore, those skilled in the art can now use these and other procedures to identify other tc homologs in other Paenibacillus species such as P. chondroitinus, P. alginolyticus, P. larvae, P. validus, P. gordonae, P alvei, P. lentimorbus, P. popilliae, P. thiaminolyticus, P. curdlanolyticus, P. kobensis, P. glucanolyticus, P. lautus, P. chibensis, P. macquariensis, P. azotofizans, P. peoriae, P. polymyxa , P. illinoisensis, P. amylolyticus, P. pabuli, and P. macerans .

Example 14

Homologs detection of the tc ORFs of IDAS 1529 in other strains of Paenibacillus by Southern Hybridization

This example illustrates how DNA fragments can be radioactively labeled as probes to investigate genomic DNA from Paenibacillus isolates for DNA sequences (preferably having some homology with the tc ORFs first detected in IDAS 1529). The results demonstrate that homologous sequences to two of the tc ROFs are detected in an isolation of Paenibacillus apairius , DB482.

Genomic DNA from various strains of Paenibacillus (or E. coli that serve as a negative control) was prepared as described above in Example 12, and digested with restriction enzymes to produce multiple fragments. A typical digestion contained 8 mg of DNA in a total volume of 400 ml of reaction buffer as supplied by the manufacturer of the enzyme EcoR I (New England Biolabs, Beverly, MA). The reaction, which contains 200 units of enzyme, was incubated overnight at 37 ° C, then placed on ice. The digested DNA was further purified and concentrated by the addition of 30 µL of 3M sodium acetate (pH 5.2) and 750 ml of ice cold 100% ice cold ethanol, followed by centrifugation. The DNA pellet was washed twice with 70% ethanol, dried under vacuum, and resuspended in 50 ml of TE buffer [10 mM Tris HCl, pH 8.0; 1 mM Ethylenediaminetetraacetic acid (EDTA)]. An aliquot was then analyzed by agarose gel electrophoresis for visual safety of borderline digestion. Similarly, DNA from cosmid SB 12 of IDAS 1529 was digested with EcoR I, and and was used as a positive control for hybridization experiments.

EcoR I digested genomic DNA fragments for transfer for Southern analysis were electrophoresed by 0.7% or 1.2% agarose gels in TEA buffer (40 mM Tris-acetate, 2 mM EDTA, pH 8, 0) (1 mg of DNA / well). In each gel, lanes containing 1 kb of 1 kb DNA Molecular Weight Scale (Invitrogen ™, Carlsbad, CA) were used to provide molecular weight size patterns. The 15 fragment sizes larger than 500 base pairs on this scale (in kilobases) are: 12.2, 11.2, 10.1, 9.2, 8.1, 7.1, 6.1, 5, 1, 4.1, 3.1, 2.0, 1.6, 1.0, 0.52, and 0.50. The DNA in the gel was stained with 50 µg / ml ethidium bromide, the gel was photographed, and then the DNA in the gel was stripped (5 min in 0.2 M HCl), denatured (15 min in 0 , 5 M NaOH, 1.5 M NaCl), was neutralized (5 min in 0.2 M HCl) and transferred to 0.45 micron MAGNA nylon transfer membrane (Osmonics, Westborough, MA) in 2 X SSC (20X SSC contains 3 M NaCl, 0.3 M sodium citrate, pH 7.0). The DNA was crosslinked with the membrane by ultraviolet light (Stratalinker®; Stratagene, La Jolla, CA) and prepared for hybridization by incubation at 60 ° C or 65 ° C for 1 to 3 hours in "Minimal Hybridization" solution [contains 10% w / v of polyethylene glycol (Molecular Weight approx. 8000), 7% w / v sodium dodecyl sulfate; 0.6 X SSC, 5 mM EDTA, 100 mg / ml AND of denatured salmon sperm, and 10 mM sodium phosphate buffer (from a 1M stock solution containing 95 g / l NaH2PO4 \ cdot1H2O and 84.5 g / l of Na2HP04 \ cdot7H2O)].

The DNA fragments of the tc ROFs for use as hybridization probes were first prepared by the Polymerase Chain Reaction (PCR) using the cosmid SB12 DNA as a template (see previous examples). The direct and reverse primers for these amplifications are listed (5 'to 3' addresses of the respective DNA strands) in Table 24, below (bases in uppercase letters correspond to protein coding regions). Primer Set One is designed to amplify, from DNA of cosmid SB 12, a DNA fragment that includes all the tc ORFs that are described as SEQ ID NO: 10, and that has some similarity with the tcaC gene of Photorhabdus (Table 6). Set Two of Primers were designed to amplify, from a fragment of the cosmid SB 12, a DNA fragment encoding the protein described as SEQ ID NO: 19. This DNA fragment and the encoded protein are somewhat longer than the tc ORF6 DNA sequence described as SEQ ID NO: 12, and the encoded protein described as SEQ ID NO: 13. The proteins described as SEQ ID NO: 13 and SEQ ID NO: 19 both had some similarity with the protein encoded by the Photorhabdus tccC gene (Table 6). The amplified PCR products were cloned into the cloning vector pCR®2.1-TOPO® (Invitrogen ™, Carlsbad, CA), and fragments containing the tc ORFs were released from the resulting clones by digestion with enzymes of restriction (listed in the Table below), followed by purification from agarose gels using the GenElute ™ Agarose Spin columns (Sigma Chemical Co, St Louis, MO). The recovered fragments were concentrated by precipitation using the Quick-Precip ™ Plus Solution according to the supplier's instructions (Edge BioSystems, Gaithersburg, MD).

TABLE 24

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Radioactively labeled DNA fragments using the High Prime Radioactive Labeling kit (Roche Diagnostics, Mannheim, Germany) according to the supplier's instructions. Unincorporated nucleotides were removed by passage to through a QIAquick® PCR Purification column (Qiagen, Inc. Valencia, CA) according to the manufacturer's instructions. He labeling of approximately 100 ng of DNA fragments by these procedures resulted in specific activities of about 0.1 µCi / ng. These AND fragments are denatured by boiling for 5 minutes, then added to the hybridization spot in hybridization solution minimum and incubated overnight at 60 ° C or 65 ° C. The emitted radioactivity was removed from the stain by rinsing to room temperature in 2 X SSC, then the radioactivity was removed  more tightly bound by washing the stain during minus one hour at 60 ° C or 65 ° C in 0.3 X SSC + 0.1% dodecyl sulfate sodium At least two washes were performed. The stain was placed on an X-ray film at -80ºC with two screens of intensification, and the exposed film was developed after 1 to 3 days of exposure. The spots were stripped of the DNA fragments hybridized by boiling for 10 minutes in 0.3 X SSC + 0.1% SDS, and reused once or twice for subsequent hybridizations.

The different fragments that hybridized to the probes derived from Sets One of the Primers and Two were observed in the genomic DNA that were obtained from strain DB482 of Paenibacillus apairius . The probe derived from Set One of Primers (primers SB126 and SB127), which detects sequences homologous to ORF5 of tc of IDAS 1529, hybridized to fragments of estimated sizes (in kilobases) of 20, 10.2, and 8.4. Within this range of molecular sizes, the mobilities of the DNA fragments can only provide estimates of the molecular sizes. The signal strength for the estimated fragments were 20 kb and 8.4 kb were much more intense than the signal strength for the estimated fragments that were 10.2 kb. Since each of these fragments is at least twice the size of the probe fragment (approximately 4.4 kb), an explanation of these results is that multiple copies of the genes that are similar to the ORF5de probes of IDAS 1529 tc , and thus, are similar to the tcaC gene of Photorhabdus , are present in the genome of strain DB482 of Paenibacillus apairius . However, other explanations for this result are possible.

The probe derived from Set Two of Primers (primers SB128 and SB129), which detects sequences homologous to the ORF6 of tc of IDAS 1529 and their flanking sequences of the 5 'end, hybridized to fragments of estimated sizes (in kilobases) of 7.8 and 4,5. The signal strength for the fragment was estimated to be 7.8 kb was much more intense than the signal strength observed for the estimated fragment that was 4.5 kb. An explanation for this result is that the Paenibacillus apairius strain DB482 had a single gene similar to the RFAS of tc IDAS 1529 and its 5 'flanking sequences and thus, is similar to the tccC gene of Photorhabdus , and that Eco RI cleaves the gene in two fragments that have unequal parts of the DNA sequences that comprise the gene. However, other explanations for this result are possible, including the presence of multiple genes with varying amounts of absolute homology with the probe.

These results (PCR amplification detection followed by DNA sequence analysis) confirm the presence of relatives of the Photorhabdus tcaC and tccC genes in the DB482 strain Paenibacillus apairius .

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Example fifteen

DB482 insecticidal activity

Paenibacillus strain strain 1529 has been shown to produce an extracellular protein that is toxic to Lepidoptera insects and has also been shown to contain a cry gene, called cry1529 . Since this strain produces an insecticide-active extracellular protein and insecticide-active intracellular proteins, the present invention includes the selection of other Paenibacillus strains for extracellular insecticidal active agents (released into the culture supernatant fluid) and / or intracellular (associated with cells). This example illustrates how fermentation broths from Paenibacillus strains can be produced, how to process these broths, and how to test samples derived from these broths to determine insecticidal activity.

15.A. Production and processing of fermentation broths of Paenibacillus

Paenibacillus strains were grown on nutrient agar plates (8 g / l nutrient broth, 15 g / l Bacto agar; Difco Laboratories, Detroit, MI) for 3-5 days at 30 ° C. a single colony was collected and inoculated in a 500 ml flask with three deflectors containing 100 ml sterile modified soy tristone broth (10-g / l tryptone, 7 g / l peptone, 3 g / l hydrolyzed soy protein, KCl 5 g / l, K 2 PO 4 2.5 g / l; Disco Laboratories, Detroit, MI). After 72 hours of incubation at 28 ° C on a 150 rpm rotary shaker, cultures were dispensed in 500 ml bottles of polyethylene and centrifuged at 4,000 xg for 45 minutes at 4 ° C. After centrifugation, the supernatant fluid was decanted and filtered through a 0.22 µm filter membrane (Millipore Corporation, Bedford, MA). The 20 X filtered culture was then concentrated using a Centricon Plus-20 centrifugation filter device with a 5,000 molecular weight cutting membrane by centrifugation at 4,000 x g. The bacterial cell pellet was resuspended in 10 mM potassium phosphate buffer (pH = 8). These samples were then tested in insect bioassay to determine the insecticidal activities contained in the processed supernatant and cell sediment samples.

15. B. Bioassay of processed supernatant insects and cell sediments

The insect species included in these trials were Diabrotica undecimpunctata howardi (South Corn Root Worm, SCR), Helicoverpa zea (Corn Cob Worm, CEW), and Heliothis virescens (Tobacco Yolk Worm, TBW ). The artificial used diet for SCR breeding and bioassay has been previously described (Rose, RL and McCabe, JM 1973. J. Econ. Entomol. 66, 398-400). Artificial diet of standard lepidoptera (Stoneville Yellow diet) was used for the breeding and bioassay of ECB, CEW, and TBW. Aliquots of 40 ul of samples from the concentrated supernatant or cell pellets were applied directly to the surface of the wells (~ 1.5 cm2) containing the artificial diet. The treated diet wells were allowed to air dry in a sterile flow hood and each well was infested with a single neonate insect incubated from surface sterilized eggs. After the test trays were sealed, they were placed in a humidified growth chamber and kept at 28 ° C for 3-5 days. The determinations of larval mortality and weight were scored afterwards. Eight insects were used per treatment.

15. C. DB482 insecticidal activity

The concentrated supernatant and cell pellets of strain DB482 had insecticidal activity against SCR, TBW, and CEW in relation to control treatments (Table 25.) It is possible that the insecticidal activity associated with concentrated supernatants and cell pellets of DB482 they are the result of two different insecticidal factors, one that is associated with cells ( that is, of the Cry type) and another that is released from the cells ( that is, from ipo CT). However, it is also possible that the insecticidal activities of both the concentrated supernatant and the cell pellets of DB482 are the result of the same insecticidal factors present in both cell fractions.

TABLE 25 DB482 insecticidal activity

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15.D. Summary of the activity selection Insecticide

This example illustrates a procedure for selecting supernatants from concentrated cultures and cell sediments from Paenibacillus strains to identify strains that possess insecticidal activity against Coleoptera and Lepidoptera insects. DB482, which is an isolation of Paenibacillus apiarius was shown herein that contained homologues of tcaA, tcaB, tcaC , and tccC . The finding of insecticidal activity in DB482 confirms that Paenibacillus strain DAS1529 is not unique within the Paenibacillus genus in relation to the production of insecticidal activities against Lepidoptera insects. Therefore, the present invention includes methods used to identify other Paenibacillus strains with insecticidal activities against Lepidoptera insects in other Paenibacillus species such as P. chondroitinus, P. alginolyticus, P. larvae, P. validus, P. gordonae, P alvei, P. lentimorbus, P. popilliae, P. thiaminolyticus, P. curdlanolyticus, P. kobensis, P. glucanolyticus, P. lautus, P. chibensis, P. macquariensis, P. azotofixans, P. peoriae, P. polymyxa , P. illinoisensis, P. amylolyticus, P. pabuli, P. macerans .

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<110> Dow AgroSciences LLC

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<120> Pesticide and polynucleotide action proteins that can be obtained from Paenibacillus species

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<130> DAS-101XC2

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<150> US 60 / 392,633

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<151> 2002-06-28

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<151> 2003-01-21

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<160> 49

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<170> PatentIn version 3.2

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         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Nucleic acid sequence of the entire insert of SB12.

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 1

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

41

42

43

44

Four. Five

46

47

48

49

fifty

51

52

53

54

55

56

57

58

59

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 2

         \ vskip0.400000 \ baselineskip
      

<211> 3264

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 2

         \ vskip1.000000 \ baselineskip
      

60

61

62

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 3

         \ vskip0.400000 \ baselineskip
      

<211> 1087

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 3

         \ vskip1.000000 \ baselineskip
      

63

64

65

66

67

68

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 4

         \ vskip0.400000 \ baselineskip
      

<211> 3618

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 4

         \ vskip1.000000 \ baselineskip
      

69

70

71

         \ vskip0.400000 \ baselineskip
      

<210> 5

         \ vskip0.400000 \ baselineskip
      

<211> 1205

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 5

72

73

74

75

76

77

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 6

         \ vskip0.400000 \ baselineskip
      

<211> 3300

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 6

         \ vskip1.000000 \ baselineskip
      

78

79

80

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 7

         \ vskip0.400000 \ baselineskip
      

<211> 1099

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 7

         \ vskip1.000000 \ baselineskip
      

81

82

83

84

85

86

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 8

         \ vskip0.400000 \ baselineskip
      

<211> 3627

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 8

         \ vskip1.000000 \ baselineskip
      

87

88

89

         \ vskip0.400000 \ baselineskip
      

<210> 9

         \ vskip0.400000 \ baselineskip
      

<211> 1208

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 9

90

91

92

93

94

95

         \ vskip0.400000 \ baselineskip
      

<210> 10

         \ vskip0.400000 \ baselineskip
      

<211> 4335

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 10

96

97

98

         \ vskip0.400000 \ baselineskip
      

<210> 11

         \ vskip0.400000 \ baselineskip
      

<211> 1444

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 11

100

101

102

103

104

105

106

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 12

         \ vskip0.400000 \ baselineskip
      

<211> 2793

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 12

107

108

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 13

         \ vskip0.400000 \ baselineskip
      

<211> 930

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 13

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

109

110

111

         \ hskip0,8cm
      

112

         \ vskip0.400000 \ baselineskip
      

<210> 14

         \ vskip0.400000 \ baselineskip
      

<211> 1791

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> ORF7 nucleic acid sequence, which encodes a Cry type protein.

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 14

113

114

         \ vskip0.400000 \ baselineskip
      

<210> 15

         \ vskip0.400000 \ baselineskip
      

<211> 596

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Coded amino acid sequence by ORF7.

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 15

         \ vskip1.000000 \ baselineskip
      

115

116

117

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 16

         \ vskip0.400000 \ baselineskip
      

<211> 1547

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Nucleic acid sequence of 16S rDNA of IDAS1529.

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 16

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

118

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<210> 17

         \ vskip0.400000 \ baselineskip
      

<211> 20

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<223> Amino acid sequence N-terminal for purified toxin fraction of broth from IDAS1529.

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 17

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

119

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 18

         \ vskip0.400000 \ baselineskip
      

<211> 379

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Amino acid sequence of thiaminase I from Bacillus thiaminolyticus .

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 18

         \ vskip1.000000 \ baselineskip
      

120

121

         \ vskip0.400000 \ baselineskip
      

<210> 19

         \ vskip0.400000 \ baselineskip
      

<211> 953

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus IDAS 1529 strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 19

122

123

124

125

         \ hskip0,8cm
      

126

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 20

         \ vskip0.400000 \ baselineskip
      

<211> 4482

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Xenorhabdus Xwi strain

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 20

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

127

128

129

         \ hskip0,8cm
      

130

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 21

         \ vskip0.400000 \ baselineskip
      

<211> 3051

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Xwi strain of Xenorhabdus

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 21

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

131

132

133

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 22

         \ vskip0.400000 \ baselineskip
      

<211> 32

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB101

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 22

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

134

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 23

         \ vskip0.400000 \ baselineskip
      

<211> 32

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB102

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 23

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

135

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 24

         \ vskip0.400000 \ baselineskip
      

<211> 28

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB103

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 24

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

136

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 25

         \ vskip0.400000 \ baselineskip
      

<211> 26

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB104

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 25

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

137

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 26

         \ vskip0.400000 \ baselineskip
      

<211> 28

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> SB105 primer

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 26

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

138

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 27

         \ vskip0.400000 \ baselineskip
      

<211> 30

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB106

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 27

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

139

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 28

         \ vskip0.400000 \ baselineskip
      

<211> 27

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB212

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_characteristic

         \ vskip0.400000 \ baselineskip
      

<222> (7) .. (7)

         \ vskip0.400000 \ baselineskip
      

<223> n = i (iosine)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 28

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

140

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 29

         \ vskip0.400000 \ baselineskip
      

<211> 25

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB213

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_characteristic

         \ vskip0.400000 \ baselineskip
      

<222> (17) .. (17)

         \ vskip0.400000 \ baselineskip
      

<223> n = i (iosine)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 29

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

141

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 30

         \ vskip0.400000 \ baselineskip
      

<211> 33

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB215

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_characteristic

         \ vskip0.400000 \ baselineskip
      

<222> (10) .. (10)

         \ vskip0.400000 \ baselineskip
      

<223> n = i (iosine)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 30

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

142

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 31

         \ vskip0.400000 \ baselineskip
      

<211> 32

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Artificial sequence

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<223> Primer SB217

         \ vskip0.400000 \ baselineskip
      

<220>

         \ vskip0.400000 \ baselineskip
      

<221> misc_characteristic

         \ vskip0.400000 \ baselineskip
      

<222> (24) .. (24)

         \ vskip0.400000 \ baselineskip
      

<223> n = i (iosine)

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 31

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,9cm
      

143

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 32

         \ vskip0.400000 \ baselineskip
      

<211> 1293

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> strain DB482 of Paenibacillus apairus

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 32

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

144

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 33

         \ vskip0.400000 \ baselineskip
      

<211> 430

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> strain DB482 of Paenibacillus apairius

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 33

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

145

146

147

         \ vskip0.400000 \ baselineskip
      

<210> 34

         \ vskip0.400000 \ baselineskip
      

<211> 340

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 34

148

         \ vskip0.400000 \ baselineskip
      

<210> 35

         \ vskip0.400000 \ baselineskip
      

<211> 565

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 35

149

         \ vskip0.400000 \ baselineskip
      

<210> 36

         \ vskip0.400000 \ baselineskip
      

<211> 113

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 36

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

150

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 37

         \ vskip0.400000 \ baselineskip
      

<211> 188

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 37

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

151

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<210> 38

         \ vskip0.400000 \ baselineskip
      

<211> 2091

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 38

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

152

         \ hskip0,9cm
      

153

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 39

         \ vskip0.400000 \ baselineskip
      

<211> 697

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 39

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,8cm
      

154

155

156

         \ hskip0,8cm
      

157

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 40

         \ vskip0.400000 \ baselineskip
      

<211> 858

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 40

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

158

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 41

         \ vskip0.400000 \ baselineskip
      

<211> 286

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Paenibacillus apairius strain DB482

         \ newpage
      

         \ vskip0.400000 \ baselineskip
      

<400> 41

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,7cm
      

159

         \ hskip0,7cm
      

160

         \ vskip0.400000 \ baselineskip
      

<210> 42

         \ vskip0.400000 \ baselineskip
      

<211> 4434

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 42

161

162

163

         \ hskip0,7cm
      

164

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 43

         \ vskip0.400000 \ baselineskip
      

<211> 4425

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 43

         \ vskip1.000000 \ baselineskip
      

165

166

167

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 44

         \ vskip0.400000 \ baselineskip
      

<211> 3132

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 44

         \ vskip1.000000 \ baselineskip
      

168

169

         \ hskip0,7cm
      

170

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 45

         \ vskip0.400000 \ baselineskip
      

<211> 2748

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 45

         \ vskip1.000000 \ baselineskip
      

         \ vskip1.000000 \ baselineskip
      

171

172

         \ hskip0,7cm
      

173

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 46

         \ vskip0.400000 \ baselineskip
      

<211> 2883

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 46

         \ vskip1.000000 \ baselineskip
      

174

175

         \ vskip0.400000 \ baselineskip
      

<210> 47

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<211> 2850

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 47

176

         \ hskip0,7cm
      

177

         \ vskip0.400000 \ baselineskip
      

<210> 48

         \ vskip0.400000 \ baselineskip
      

<211> 2817

         \ vskip0.400000 \ baselineskip
      

<212> DNA

         \ vskip0.400000 \ baselineskip
      

<213> Photorhabdus strain W14

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 48

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,7cm
      

178

179

         \ hskip0,7cm
      

180

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<210> 49

         \ vskip0.400000 \ baselineskip
      

<211> 2538

         \ vskip0.400000 \ baselineskip
      

<212> PRT

         \ vskip0.400000 \ baselineskip
      

<213> Xenorhabdus nematophilus

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.400000 \ baselineskip
      

<400> 49

         \ vskip1.000000 \ baselineskip
      

         \ hskip0,7cm
      

181

182

183

184

185

186

187

188

189

190

191

192

Claims (3)

1. A method of selecting a Paenibacillus isolation culture for a gene encoding a protein selected from the group consisting of a toxin complex protein, wherein said method comprises at least one of the following steps: (a) obtain DNA from said culture and test said DNA to evaluate the presence of said gene; Y (b) obtain protein produced by said culture and test said protein to evaluate the presence of a protein which indicates the presence of a protein that indicates the presence of said gene in said isolation, and at least one of the following stages: (c) perform the chain reaction of the polymerase (PCR) with at least one primer that hybridizes to PCR conditions with a polynucleotide encoding a amino acid sequence selected from the group constituted by SEQ ID NO: 13 and SEQ ID NO: 41, (d) immunoreaction of an antibody with said protein in which said antibody binds to a sequence of amino acids selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 41, e (e) hybridize a nucleic acid probe with said DNA in which said probe hybridizes under conditions of moderate to high stringency with a polynucleotide encoding a amino acid sequence selected from the group constituted by SEQ ID NO: 13 and SEQ ID NO: 41. 2. A protein that has toxin activity against an insect in which a sequence of polynucleotides that encodes said protein to hybridize under stringent conditions moderate to high with the complement of an acid sequence nucleic encoding a selected amino acid sequence between the group consisting of SEQ ID NO: 13 and SEQ ID NO: 41. 3. An insect control procedure in which said procedure comprises the step of contacting said insect with a protein that has at least 90% identity with an amino acid sequence selected from the group constituted by SEQ ID numbers: 13, and 41.